JP7128819B2 - ADAM9 binding molecules, and methods of use thereof - Google Patents

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Patent Application Serial No. 62/438,516 (filed December 23, 2016; pending), which is incorporated herein by reference in its entirety. be
SEQUENCE LISTING REFERENCE

37 C.F.R., disclosed on a computer readable medium (file name: 1301.0147PCT_Sequence_Listing_ST25.txt, dated November 29, 2017, having a size of 175,962 bytes). F. R. 1.821 et seq. and contains one or more Sequence Listings pursuant to , which file is hereby incorporated by reference in its entirety.

The present invention provides molecules, such as monospecific and bispecific antibodies, including diabodies, BiTEs, and antibodies capable of specifically binding to "Disintegrin and Metalloproteinase Domain-Containing Protein 9" ("ADAM9"). It relates to specific, trispecific or multispecific binding molecules. The invention particularly relates to binding molecules capable of exhibiting high affinity binding to human and non-human ADAM9. The invention more particularly relates to molecules thereby cross-reacting with human ADAM9 and non-human primate (eg, cynomolgus monkey) ADAM9. The invention further provides light chain variable (VL) domains and/or heavy chain variable humanized and/or deimmunized to exhibit reduced immunogenicity upon administration of such ADAM9 binding molecules to a recipient subject. All ADAM9-binding molecules containing a (VH) domain. The invention also relates to pharmaceutical compositions comprising any such ADAM9 binding molecules, and methods comprising the use of any such ADAM9 binding molecules in the treatment of cancer and other diseases and conditions.

ADAMs are a family of proteins involved in various physiological and pathological processes (Amendola, R.S. et al. (2015) “ADAM9 Disintegrin Domain Activates Human Neutrophils Through An Autocrine Circuit Involving Integrins And CXCR2,” J. Leukocyte Biol. 97(5):951-962; Edwars, D.R. et al. (2008) "The ADAM Metalloproteases," Molec. Aspects Med. 29:258-289). At least 40 gene members of the family have been identified, and at least 21 of such members are believed to be functional in humans (Li, J. et al. (2016) “Overexpression of ADAM9 Promotes Colon Cancer Cells Invasion,” J. Invest. Surg. 26(3):127-133; Duffy, M.J. et al. (2011) “The ADAMs Family Of Proteases: New Biomarkers And Therapeutic Targets For Cancer?,” Clin. 8:9:1-13; see also U.S. Patent Publication No. 2013/0045244).

ADAM family members have a well-conserved structure with eight domains, including a metalloprotease domain and an integrin-binding (disintegrin) domain (Duffy, M.J. et al. (2009) “The Role Of ADAMs In Disease Pathophysiology," Clin. Chim. Acta 403:31-36). ADAM metalloprotease domains have been reported to act as sheddases and regulate a range of biological processes by cleaving transmembrane proteins, which in turn can act as soluble ligands. , and can modulate cell signaling (Amendola, R.S. et al. (2015) “ADAM9 Disintegrin Domain Activates Human Neutrophils Through An Autocrine Circuit Involving Integrins And CXCR2,” J. Leukocyte Biol. 97(5):951-962; Ito, N. et al. (2004) "ADAMs, A Disintegrin And Metalloproteinases, Mediate Shedding Of Oxytocinase," Biochem. Biophys. Res. Commun. 314 (2004) 1008–1013).

ADAM9 is a member of the ADAM family of molecules. It is synthesized as an inactive form that is proteolytically cleaved to produce the active enzyme. Processing at upstream sites is particularly important for proenzyme activation. ADAM9 is a fibroblast (Zigrino, P. et al. (2011) “The Disintegrin-Like And Cysteine-Rich Domains Of ADAM-9 Mediate Interactions Between Melanoma Cells And Fibroblasts,” J. Biol. Chem. 286:6801-6807 ), activated vascular smooth muscle cells (Sun, C. et al. (2010) “ADAM15 Regulates Endothelial Permeability And Neutrophil Migration Via Src/ERK1/2 Signaling,” Cardiovasc. Res. 87:348-355), monocytes ( Namba, K. et al. (2001) “Involvement Of ADAM9 In Multinucleated Giant Cell Formation Of Blood Monocytes,” Cell. Immunol. 213:104-113), activated macrophages (Oksala, N. et al. (2009) “ ADAM-9, ADAM-15, And ADAM-17 Are Upregulated In Macrophages In Advanced Human Atherosclerotic Plaques In Aorta And Carotid And Femoral Arteries – Tampere Vascular Study,” Ann. Med. 41:279-290). be.

The metalloprotease activity of ADAM9 is involved in the degradation of matrix components, thereby enabling tumor cell migration (Amendola, R.S. et al. (2015) “ADAM9 Disintegrin Domain Activates Human Neutrophils Through An Autocrine Circuit Involving Integrins And CXCR2 ,” J. Leukocyte Biol. 97(5):951-962). Its disintegrin domain, which is highly homologous to many snake venom disintegrins, allows interaction between ADAM9 and integrins, allowing ADAM9 to positively or negatively regulate cell adhesion events ( Zigrino, P. et al. (2011) "The Disintegrin-Like And Cysteine-Rich Domains Of ADAM-9 Mediate Interactions Between Melanoma Cells And Fibroblasts," J. Biol. Chem. 286:6801-6807; Karadag, A. et al. al. (2006) “ADAM-9 (MDC-9/Meltringamma), A Member Of The A Disintegrin And Metalloproteinase Family, Regulates Myeloma-Cell-Induced Interleukin-6 Production In Osteoblasts By Direct Interaction With The Alpha(v)Beta5 Integrin et al. (2009) "Inhibition Of Platelets And Tumor Cell Adhesion By The Disintegrin Domain Of Human ADAM9 To Collagen I Under Dynamic Flow Conditions," Biochimie 91:1045-1052). The ADAM9 disintegrin domain has been shown to interact with α6β1, α6β4, αvβ5 and α9β1 integrins.

Expression of ADAM9 has been found to be associated with disease, particularly cancer. ADAM9 has been found to cleave and release many molecules that have important roles in tumorigenesis and angiogenesis, including TEK, KDR, EPHB4, CD40, VCAM1 and CDH5. ADAM9 is regulated by many types of tumor cells, including breast, colon, gastric, glioma, liver, non-small cell lung, melanoma, myeloma, pancreatic and prostate cancer tumor cells. (Yoshimasu, T. et al. (2004) “Overexpression Of ADAM9 In Non-Small Cell Lung Cancer Correlates With Brain Metastasis,” Cancer Res. 64:4190-4196; Peduto, L. et al. (2005) "Critical Function For ADAM9 In Mouse Prostate Cancer," Cancer Res. 65:9312-9319; Zigrino, P. et al. (2005) "ADAM-9 Expression And Regulation In Human Skin Melanoma And Melanoma Cell Lines," Int. J. Cancer 116:853-859; Fritzsche, F.R. et al. (2008) “ADAM9 Is Highly Expressed In Renal Cell Cancer And Is Associated With Tumor Progression,” BMC Cancer 8:179:1-9; Fry, J.L. et al. (2010) “Secreted And Membrane-Bound Isoforms Of Protease ADAM9 Have Opposing Effects On Breast Cancer Cell Migration,” Cancer Res. 70, 8187-8198; Chang, L. et al. (2016) “Combined Rnai Targeting Human Stat3 And ADAM9 As Gene Therapy For Non-Small Cell Lung Cancer,” Oncology Letters 11:1242-1250; Fan, X. et al. (2016) “ADAM9 Expression Is Associate with Glioma Tu Mor Grade and Histological Type, and Acts as a Prognostic Factor in Lower-Grade Gliomas,” Int. J. Mol. Sci. 17:1276:1-11).

Importantly, increased ADAM9 expression has been found to positively correlate with tumor grade and metastatic potential (Amendola, R.S. et al. (2015) “ADAM9 Disintegrin Domain Activates Human Neutrophils Through An Autocrine Circuit Involving Integrins And CXCR2,” J. Leukocyte Biol. 97(5):951-962; Fan, X. et al. (2016) “ADAM9 Expression Is Associate with Glioma Tumor Grade and Histological Type, and Acts as a Prognostic Factor in Lower- Grade Gliomas,” Int. J. Mol. Sci. 17:1276:1-11; Li, J. et al. (2016) “Overexpression of ADAM9 Promotes Colon Cancer Cells Invasion,” J. Invest. Surg. 26(3 ): 127-133). Moreover, ADAM9 and its secreted soluble isoforms appear to be important for cancer cell dissemination (Amendola, R.S. et al. (2015) “ADAM9 Disintegrin Domain Activates Human Neutrophils Through An Autocrine Circuit Involving Integrins And CXCR2,” J. Leukocyte Biol. 97(5):951-962; Fry, J.L. et al. (2010) “Secreted And Membrane-Bound Isoforms Of Protease ADAM9 Have Opposing Effects On Breast Cancer Cell Migration,” Cancer Res. 70 , 8187–8198; Mazzocca, A. (2005) "A Secreted Form Of ADAM9 Promotes Carcinoma Invasion Through Tumor-Stromal Interactions," Cancer Res. 65:4728-4738; 7,585,634; 7,829,277; 8,101,361; and 8,445,198 and U.S. Patent Publication No. 2009/0023149).

Therefore, many studies have identified ADAM9 as a potential target for anticancer therapy (Peduto, L. (2009) “ADAM9 As A Potential Target Molecule In Cancer,” Curr. Pharm. Des. 15:2282- 2287; Duffy, M.J. et al. (2009) "Role Of ADAMs In Cancer Formation And Progression," Clin. Cancer Res. 15:1140-1144; Duffy, M.J. et al. Biomarkers And Therapeutic Targets For Cancer?” Clin. Proteomics 8:9:1-13; Josson, S. et al. Prostate 71(3):232-240; U.S. Patent Publication Nos. 2016/0138113, 2016/0068909, 2016/0024582, 2015/0368352, 2015/0337356, 2015 /0337048, 2015/0010575, 2014/0342946, 2012/0077694, 2011/0151536, 2011/0129450, 2010/0291063, 2010/ 0233079, 2010/0112713, 2009/0285840, 2009/0203051, 2004/0092466, 2003/0091568, and 2002/0068062, and PCT Publication Nos. WO2016/077505, WO2014/205293, WO2014/186364, WO2014/124326, WO2014/108480 , WO2013/119960, WO2013/098797, WO2013/049704, and WO2011/100362). In addition, ADAM9 expression has also been found to be associated with lung disease and inflammation (e.g., U.S. Patent Publication Nos. 2016/0068909; 2012/0149595; 2009/0233300; 2006). /0270618; and 2009/0142301). Antibodies that bind ADAM9 are commercially available from Abcam, Thermofisher, Sigma-Aldrich and other companies.

However, despite all past progress, there remains a need for high-affinity ADAM9-binding molecules that exhibit minimal binding to normal tissues and are capable of binding human and non-human ADAM9 with similar high affinity. It is The present invention addresses this need and the need for improved treatments for cancer.

The present invention provides monospecific and bispecific, trispecific antibodies, including antibodies capable of specifically binding diabodies, BiTEs, and "disintegrin and metalloproteinase domain-containing protein 9" ("ADAM9"). or molecules such as multispecific binding molecules. The invention particularly relates to binding molecules capable of exhibiting high affinity binding to human and non-human ADAM9. The invention is more particularly directed to molecules thereby cross-reactive with human ADAM9 and non-human primate (eg, cynomolgus monkey) ADAM9. The invention further provides light chain variable (VL) domains that are humanized and/or deimmunized to exhibit reduced immunogenicity upon administration of such ADAM9 binding molecules to a recipient subject and /or to all such ADAM9 binding molecules comprising a heavy chain variable (VH) domain. The present invention is also directed to pharmaceutical compositions comprising any such ADAM9 binding molecules, methods comprising using any such ADAM9 binding molecules in the treatment of cancer and other diseases and conditions. do.

In particular, the invention provides ADAM9 binding molecules comprising ADAM9 binding domains, such ADAM9 binding domains comprising light chain variable (VL) domains and heavy chain variable (VH) domains, such heavy chain variable Domains include CDR _H 1, CDR _H 2 and CDR _H 3 domains, such light chain variable domains include CDR _L 1, CDR _L 2 and CDR _L 3 domains:
(A) Such CDR _H 1, CDR _H 2 and CDR _H 3 domains are the CDR _H 1, CDR _H 2 and CDRs of the heavy chain variable (VH) domain of optimized variants of MAB-A. such _{CDR L} 1 domain, CDR _L 2 domain, and CDR _L 3 domain are the CDR _L 1 domain, CDR _L 2 domain of the light chain variable (VL) domain of MAB-A; or (B) such CDR _H 1, CDR _H 2 and _{CDR H} 3 domains are the CDR _H of the heavy chain variable (VH) domain of MAB-A 1 domain, CDR _H 2 domain and CDR _H 3 domain; and such CDR _L 1 domain, CDR _L 2 domain and CDR _L 3 domain are the light or ( _C ) such CDR _H 1, _{CDR H} 2 and _{CDR H} 3 domains are , the amino acid sequences of the CDR _H 1, CDR _H 2 and CDR _H 3 domains of the heavy chain variable (VH) domain of an optimized variant of MAB-A; and such CDR _L 1 domain, CDR _L 2, and CDR _L 3 domains have the amino acid sequences of the CDR _L 1, CDR _L 2, and CDR _L 3 domains of the light chain variable (VL) domain of an optimized variant of MAB-A.

The invention specifically provides that such ADAM9 binding domains:
(A) (1) CDR _H 1, CDR _H 2 and CDR _H 3 domains of the heavy chain variable (VH) domain of MAB-A; and (2) FR1 of the VH domain of a humanized variant of MAB-A. , FR2, FR3 and FR4; or (B) (1) the CDR _L 1, CDR _L 2 and CDR _L 3 domains of the light chain variable (VL) domain of MAB-A; and (2) human of MAB-A or (C)(1) CDR _H 1 domain, CDR _H 2 domain and CDRs of the heavy chain variable (VH) domain of an optimized variant of MAB-A. and (2) FR1, FR2, FR3 and FR4 of the _VH domain of a humanized variant of MAB-A; or (D) (1) the light chain variable (VL) of an optimized variant of MAB-A. ₍ 2) FR1, FR2, FR3 and FR4 of the _VL domain of the humanized variant of MAB-A; or (E) (1) _MAB- (2) VL light chain variable (VL) domain of a humanized/optimized variant of MAB-A; Regarding morphology.

The invention further provides that such heavy chain variable (VH) domain of such optimized variants of MAB-A has SEQ ID NO: 15:
EVQLVESGGG LVKPGGSLRL SCAAS GFTFS SYWX _1H WVRQA
PGKGLEWVG E IIPIX ₂ GHTNY NEX ₃ FX ₄ X ₅ RFTI SLDNSKNTLY
LQMGSLRAED TAVYYCAR GG YYYYX ₆ X ₇ X ₈ X ₉ X ₁₀ X ₁₁
DYWGQGTTVT VSS
containing the amino acid sequence of
wherein: X ₁ , X ₂ , X ₃ , X ₄ , X ₅ and X ₆ are independently selected;
X ₁ is M or I; X ₂ is N or F; _{X 3} is K or R; _{X 4} is K or Q; is F, Y, W, I, L, V, T, G or D;
X ₇ , X ₈ , X ₉ , X ₁₀ and X ₁₁ are:
X ₇ is K or R; X ₈ is F or M; X ₉ is G; _{X 10 is} W or F; X ₁₁ is M, L or K;
X ₇ is N or H; X ₈ is S or K; _{X 9 is} G or A; X ₁₀ is T or V; X ₁₁ is M, L or K;
X ₇ is G; X ₈ is K; _{X 9 is} G or A; X ₁₀ is V; X ₁₁ is M, L or K;
X ₇ is G; X ₈ is K, M or N; X ₉ is G; _{X 10 is} V or T; X ₁₁ is L or M;
X ₇ is G; X ₈ is S; X ₉ is G; _{X 10 is} V; X ₁₁ is L;
X ₇ is S; X ₈ is N; X ₉ is A; _{X 10 is} V; X ₁₁ is L
All such ADAM9 binding molecule embodiments are selected to be

The invention further provides that such CDR _H 1, CDR _H 2 and CDR _H 3 domains of such heavy chain variable (VH) domains of such optimized variants of MAB-A each comprise:
(1) SEQ ID NO: 47 (SYWX ₁ H)
where: X ₁ is M or I;
_{( 2} ) SEQ ID NO: 48 _{( EIIPIX2GHTNYNEX3FX4X5} )
wherein: X ₂ , X ₃ , X ₄ and X ₅ are independently selected;
X ₂ is N or F _; X ₃ is K or R _; X ₄ is K or Q; and X ₅ is S or G; ₈ X ₉ X ₁₀ X ₁₁ DY)
wherein: X ₆ is P, F, Y, W, I, L, V, T, G or D and X ₇ , X ₈ , X ₉ , X ₁₀ and X ₁₁ are:
(A) If _X6 is P:
X ₇ is K or R; X ₈ is F or M; X ₉ is G; X ₁₀ is W or F; and X ₁₁ is M, L or K;
(B) when _X6 is F, Y or W:
X ₇ is N or H; X ₈ is S or K; X ₉ is G or A; X ₁₀ is T or V; and X ₁₁ is M, L or K;
(C) When X6 is I, L or _V :
X ₇ is G; X ₈ is K; X ₉ is G or A; X ₁₀ is V; and X ₁₁ is M, L or K;
(D) If _X6 is T:
X ₇ is G; X ₈ is K, M or N; X ₉ is G; X ₁₀ is V or T; and X ₁₁ is L or M;
(E) If _X6 is G:
X ₇ is G; X ₈ is S; X ₉ is G; X ₁₀ is _V ; and X ₁₁ is L;
X ₇ is S; X ₈ is N; X ₉ is A; X ₁₀ is V; and X ₁₁ is L
All such ADAM9 binding molecule embodiments having an amino acid sequence of

The invention further provides that such heavy chain variable (VH) domains of such optimized variants of MAB-A are:
(1) hMAB-A VH(1) (SEQ ID NO: 16);
(2) hMAB-A VH(2) (SEQ ID NO: 17);
(3) hMAB-A VH(3) (SEQ ID NO: 18);
(4) hMAB-A VH(4) (SEQ ID NO: 19);
(5) hMAB-A VH(2A) (SEQ ID NO: 20);
(6) hMAB-A VH(2B) (SEQ ID NO:21);
(7) hMAB-A VH(2C) (SEQ ID NO:22);
(8) hMAB-A VH(2D) (SEQ ID NO:23);
(9) hMAB-A VH(2E) (SEQ ID NO:24);
(10) hMAB-A VH(2F) (SEQ ID NO: 25);
(11) hMAB-A VH(2G) (SEQ ID NO:26);
(12) hMAB-A VH(2H) (SEQ ID NO:27);
(13) hMAB-A VH(2I) (SEQ ID NO:28); and (14) hMAB-A VH(2J) (SEQ ID NO:29)
Embodiments of all such ADAM9 binding molecules selected from the group consisting of:

The invention further provides that such light chain variable (VL) domain has the amino acid sequence of SEQ ID NO:53:
DIV MTQ SPDS LAVS LGERAT ISC X ₁₂ AS QSVD
YX ₁₃ GDSYX ₁₄ N WY QQKPGQPPKL LIY AASDLES
GIPARFSGSG SGTDFTLTIS SLEPEDFATY
YC QQSX ₁₅ X ₁₆ X ₁₇ PF T FGQGTKLEI K
wherein: _X12 , _X13 , _X14 , _X15 , _X16 , and _X17 are independently selected, and _X12 is K or R; _X13 is D or S X ₁₄ is M or L; X ₁₅ is H or Y; X ₁₆ is E or S; and X ₁₇ is D or T.
All such ADAM9 binding molecule embodiments, including amino acid sequences.

The invention further provides that such CDR _L 1, CDR _L 2 and CDR _L 3 domains of such light chain variable (VL) domains of such optimized variants of MAB-A are each:
( ₁ ) SEQ ID NO: 66 _{( X12ASQSVDYX13GDSYX14N} )
wherein: X ₁₂ , X ₁₃ , X ₁₄ are independently selected, and X ₁₂ is K or R; X ₁₃ is D or S; and X ₁₄ is M or L;
(2) SEQ ID NO: 13 (AASDLES); and (3) SEQ ID NO: 67 (QQSX ₁₅ X ₁₆ X ₁₇ PFT)
X ₁₅ is H or _Y ; _{X 16 is} E or S; and X ₁₇ is D or _T. with respect to all such ADAM9 binding molecule embodiments.

The invention further provides that such light chain variable (VL) domains of such optimized variants of MAB-A are:
(1) hMAB-A VL(1) (SEQ ID NO:54);
(2) hMAB-A VL(2) (SEQ ID NO:55);
(3) hMAB-A VL(3) (SEQ ID NO:56);
(4) hMAB-A VL(4) (SEQ ID NO:57);
(5) hMAB-A VL(2A) (SEQ ID NO: 20)
Embodiments of such ADAM9 binding molecules selected from the group consisting of:

The invention further provides that the ADAM9 binding domain is:
(A) (1) a CDR _H 1 domain comprising the amino acid sequence SYWMH (SEQ ID NO: 8);
(2) a CDR _H2 domain comprising the amino acid sequence EIIPIFGHTNYNEKFKS (SEQ ID NO:35); or (3) a CDR _H3 domain comprising the amino acid sequence GGYYYYPRQGFLDY (SEQ ID NO:45);
or (B) (1) a CDR _L 1 domain comprising the amino acid sequence KASQSVDYSGDSYMN (SEQ ID NO: 62);
(2) a CDR _L2 domain comprising the amino acid sequence AASDLES (SEQ ID NO:13); or (3) a CDR _L3 domain comprising the amino acid sequence QQSHEDPFT (SEQ ID NO:14);
Embodiments of such ADAM9 binding molecules comprising:

The invention further provides that the ADAM9 binding domain comprises a CDR _H 1 domain comprising the amino acid sequence SYWMH (SEQ ID NO: 8), a CDR _H 2 domain comprising the amino acid sequence EIIPIFGHTNYNEKFKS (SEQ ID NO: 35) and an amino acid sequence GGYYYYPRQGFLDY (SEQ ID NO: 45). and a CDR _H3 domain comprising

The invention further provides that the ADAM9 binding domain comprises a CDR _L 1 domain comprising the amino acid sequence KASQSVDYSGDSYMN (SEQ ID NO: 62), a CDR _L 2 domain comprising the amino acid sequence AASDLES (SEQ ID NO: 13) and an amino acid sequence QQSHEDPFT (SEQ ID NO: 14). and a CDR _L 3 domain comprising

The invention further provides that such ADAM9 binding domains:
(A) the heavy chain variable (VH) domain of hMAB-A(2I.2) (SEQ ID NO:28); or (B) the light chain variable (VL) domain of hMAB-A(2I.2) (SEQ ID NO:55) or (C) the heavy chain variable (VH) domain of hMAB-A(2I.2) (SEQ ID NO:28) and the light chain variable (VL) domain of hMAB-A(2I.2) (SEQ ID NO:55);
Embodiments of such ADAM9 binding molecules, comprising:

The invention further provides that such ADAM9 binding domains have (a) SEQ ID NOs: 8, 35 and 10 and SEQ ID NOs: 62, 13 and 14, respectively;
(b) SEQ ID NOs: 8, 35 and 10 and SEQ ID NOs: 63, 13 and 14, respectively;
(c) SEQ ID NOs: 8, 36 and 10 and SEQ ID NOs: 63, 13 and 14, respectively; and (d) SEQ ID NOs: 34, 36 and 10 and SEQ ID NOs: 64, 13 and 65
Such ADAM9 binding comprising a CDR _H 1 domain, a CDR _H 2 domain, and a CDR _H 3 domain and a CDR _L 1 domain, a CDR _L 2 domain, and a CDR _L 3 domain, having a sequence selected from the group consisting of It relates to molecular embodiments.

The invention further provides that such ADAM9 binding domains:
(a) SEQ ID NO: 17 and SEQ ID NO: 55, respectively;
(b) SEQ ID NO: 17 and SEQ ID NO: 56, respectively;
(c) SEQ ID NO: 18 and SEQ ID NO: 56, respectively; and (d) SEQ ID NO: 19 and SEQ ID NO: 57, respectively
ADAM9 binding comprising a heavy chain variable domain (VH) and a light chain variable domain (VL) having a sequence that is at least 90%, at least 95%, or at least 99% identical to a sequence selected from the group consisting of It relates to molecular embodiments.

The invention further provides that such ADAM9 binding domains:
(a) SEQ ID NO: 17 and SEQ ID NO: 55, respectively;
(b) SEQ ID NO: 17 and SEQ ID NO: 56, respectively;
(c) SEQ ID NO: 18 and SEQ ID NO: 56, respectively; and (d) SEQ ID NO: 19 and SEQ ID NO: 57, respectively
ADAM9 binding molecule embodiments comprising a heavy chain variable domain (VH) and a light chain variable domain (VL) having a sequence selected from the group consisting of:

The invention further provides that such ADAM9 binding domains have at least a 150-fold enhancement in binding affinity to cynomolgus monkey ADAM9 compared to MAB-A and retain high affinity binding to human ADAM9. to embodiments of ADAM9 binding molecules.

The invention further provides that such ADAM9 binding domains:
(a) SEQ ID NOs: 8, 35 and 37 and SEQ ID NOs: 62, 13 and 14, respectively;
(b) SEQ ID NOs: 8, 35 and 38 and SEQ ID NOs: 62, 13 and 14, respectively;
(c) SEQ ID NOs: 8, 35 and 39 and SEQ ID NOs: 62, 13 and 14, respectively;
(d) SEQ ID NOs: 8, 35 and 40 and SEQ ID NOs: 62, 13 and 14, respectively;
(e) SEQ ID NOs: 8, 35 and 41 and SEQ ID NOs: 62, 13 and 14, respectively;
(f) SEQ ID NOs: 8, 35 and 42 and SEQ ID NOs: 62, 13 and 14, respectively;
(g) SEQ ID NOs: 8, 35 and 43 and SEQ ID NOs: 62, 13 and 14, respectively;
(h) SEQ ID NOs: 8, 35 and 44 and SEQ ID NOs: 62, 13 and 14, respectively;
(i) SEQ ID NOs:8, 35 and 45 and SEQ ID NOs:62, 13 and 14 respectively; and (j) SEQ ID NOs:8, 35 and 46 and SEQ ID NOs:62, 13 and 14 respectively
Embodiments of ADAM9-binding molecules comprising CDR _H 1, CDR _H 2, and CDR _H 3 domains and CDR _L 1, CDR _L 2, and CDR _L 3 domains having a sequence selected from the group consisting of Regarding.

The invention further provides that such ADAM9 binding domains:
(a) SEQ ID NO: 20 and SEQ ID NO: 55, respectively;
(b) SEQ ID NO: 21 and SEQ ID NO: 55, respectively;
(c) SEQ ID NO: 22 and SEQ ID NO: 55, respectively;
(d) SEQ ID NO: 23 and SEQ ID NO: 55, respectively;
(e) SEQ ID NO: 24 and SEQ ID NO: 55, respectively;
(f) SEQ ID NO: 25 and SEQ ID NO: 55, respectively;
(g) SEQ ID NO: 26 and SEQ ID NO: 55, respectively;
(h) SEQ ID NO: 27 and SEQ ID NO: 55, respectively;
(i) SEQ ID NO:28 and SEQ ID NO:55, respectively; and (j) SEQ ID NO:29 and SEQ ID NO:55, respectively
heavy chain variable domains (VH) and light chain variable domains (VL) having sequences that are at least 90%, at least 95%, or at least 99% identical to a sequence selected from the group consisting of Embodiments of ADAM9 binding molecules.

The invention further provides that such ADAM9 binding domains:
(a) SEQ ID NO: 20 and SEQ ID NO: 55, respectively;
(b) SEQ ID NO: 21 and SEQ ID NO: 55, respectively;
(c) SEQ ID NO: 22 and SEQ ID NO: 55, respectively;
(d) SEQ ID NO: 23 and SEQ ID NO: 55, respectively;
(e) SEQ ID NO: 24 and SEQ ID NO: 55, respectively;
(f) SEQ ID NO: 25 and SEQ ID NO: 55, respectively;
(g) SEQ ID NO: 26 and SEQ ID NO: 55, respectively;
(h) SEQ ID NO: 27 and SEQ ID NO: 55, respectively;
(i) SEQ ID NO:28 and SEQ ID NO:55, respectively; and (j) SEQ ID NO:29 and SEQ ID NO:55, respectively
ADAM9 binding molecule embodiments comprising a heavy chain variable domain (VH) and a light chain variable domain (VL) having a sequence selected from the group consisting of:

The invention further provides for the practice of all such ADAM9 binding molecules, wherein such molecules are monospecific ADAM9 binding antibodies or ADAM9 binding fragments thereof, or such molecules are bispecific antibodies. Regarding morphology.

The invention further provides all such ADAM9 binding molecules, wherein such molecules are diabodies, wherein such diabodies are covalently linked complexes comprising 2, 3, 4 or 5 polypeptide chains. with respect to an embodiment of

The present invention further extends to all such molecules are trivalent binding molecules, and wherein such trivalent binding molecules are covalently linked complexes comprising 3, 4, 5, or more polypeptide chains. Embodiments of such ADAM9 binding molecules are provided.

The invention further relates to embodiments of such ADAM9 binding molecules, wherein such molecules comprise an albumin binding domain (ABD).

The present invention further relates to embodiments of such ADAM9 binding molecules, wherein such molecules comprise an Fc region, in particular such Fc regions are:
(a) one or more amino acid modifications that decrease the affinity of the variant Fc region of the FcγR; and/or (b) one or more amino acid modifications that enhance the serum half-life of such ADAM9 binding molecules. It relates to embodiments that are regions.

The invention further provides that one or more such amino acid modifications that reduce the affinity of the FcγR variant Fc region are:
(A) L234A;
(B) L235A; or (C) L234A and L235A
includes;
Here, such numbering is of the EU index as in Kabat, relating to embodiments of ADAM9 binding molecules.

The invention further provides that one or more such amino acid modifications that enhance the serum half-life of such ADAM9 binding molecules are:
(A) M252Y;
(B) M252Y and S254T;
(C) M252Y and T256E;
(D) M252Y, S254T and T256E; or (E) K288D and H435K
includes;
Here, such numbering relates to embodiments of ADAM9 binding molecules that are of the EU index as in Kabat.

The present invention further provides that such molecules are bispecific, comprising an epitope binding site capable of immunospecific binding to an epitope of ADAM9 and an epitope of the molecule present on the surface of effector cells. Embodiments of ADAM9 binding molecules comprising an epitope binding site capable of binding functionally.

The invention further provides that such a molecule has two epitope binding sites capable of immunospecific binding to epitope(s) of ADAM9 and epitope(s) of the molecule present on the surface of effector cells. An embodiment of an ADAM9 binding molecule comprising two epitope binding sites capable of immunospecific binding to.

The invention further provides that such molecules are trispecific:
(a) one epitope binding site capable of immunospecific binding to an epitope of ADAM9;
(b) one epitope binding site capable of immunospecific binding to an epitope of a molecule present on the surface of the first effector cell;
(c) one epitope binding site capable of immunospecific binding to an epitope of a second molecule present on the surface of an effector cell;
Embodiments of ADAM9 binding molecules comprising:

The invention further relates to embodiments of ADAM9-binding molecules, wherein such molecules are capable of simultaneously binding ADAM9 and such molecules present on the surface of effector cells.

The invention further relates to embodiments of ADAM9-binding molecules, wherein such molecules deposited on the surface of effector cells are CD2, CD3, CD8, TCR, or NKG2D.

The invention further relates to embodiments of ADAM9 binding molecules, wherein such effector cells are cytotoxic T cells or natural killer (NK) cells.

The present invention further relates to embodiments of ADAM9 binding molecules, wherein said first such molecule present on the surface of effector cells is CD3 and said second such molecule present on the surface of effector cells is CD8. .

The invention further relates to embodiments of ADAM9-binding molecules, wherein such ADAM9-binding molecules mediate coordinated binding of ADAM9-expressing cells and cytotoxic T cells.

The invention further relates to pharmaceutical compositions comprising an effective amount of any of the ADAM9 binding molecules described above and a pharmaceutically acceptable carrier, excipient or diluent.

The invention further relates to the use of any of said ADAM9 binding molecules or of said pharmaceutical compositions in the treatment of diseases or conditions associated with or characterized by expression of ADAM9.

The present invention is particularly directed to the disease or condition associated with or characterized by the expression of ADAM9 is cancer, especially such cancers are: bladder cancer, breast cancer, cervical cancer, colorectal cancer Cancer (especially adenocarcinoma, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, primary colon lymphoma, leiomyosarcoma, melanoma, or squamous cell carcinoma), esophageal cancer, stomach cancer, head and neck cancer, liver cancer cancer, non-small cell lung cancer (especially squamous cell carcinoma, adenocarcinoma, or undifferentiated large cell carcinoma), bone marrow cancer, ovarian cancer, pancreatic cancer, prostate cancer, renal cell cancer, thyroid cancer, A use selected from the group consisting of testicular cancer and uterine cancer.

The invention further provides a method for treating a disease or condition associated with or characterized by ADAM9 expression in a subject, comprising administering to such subject an effective amount of said ADAM9-binding molecule. or a method comprising administering any of the aforementioned pharmaceutical compositions.

The present invention particularly relates to methods wherein the disease or condition associated with or characterized by the expression of ADAM9 is cancer, particularly such cancers are: bladder cancer, breast cancer, cervical cancer, colon cancer. rectal cancer (especially adenocarcinoma, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, primary colon lymphoma, leiomyosarcoma, melanoma, or squamous cell carcinoma), esophageal cancer, stomach cancer, head and neck cancer, liver cancer cancer, non-small cell lung cancer (especially squamous cell carcinoma, adenocarcinoma, or undifferentiated large cell carcinoma), bone marrow cancer, ovarian cancer, pancreatic cancer, prostate cancer, renal cell cancer, thyroid cancer, testicular cancer A method selected from the group consisting of cancer and uterine cancer.

FIG. 1 shows a representative covalent with two epitope binding sites composed of two polypeptide chains, each with an E-coil or K-coil heterodimer-promoting domain (alternative heterodimer-promoting domains are presented below). Schematic representation of bound diabody is provided. Cysteine residues may be present in the linker and/or in the heterodimer promoting domain as shown in Figure 3B. VL and VH domains that recognize the same epitope are indicated using the same shading or fill pattern. FIG. 2 depicts a representative covalent linkage with two epitope binding sites composed of two polypeptide chains, each with CH2 and CH3 domains, such that the linked chains form all or part of an Fc region. FIG. 1 provides a schematic representation of a diabody molecule. VL and VH domains that recognize the same epitope are indicated using the same shading or fill pattern. Figures 3A-3C provide a schematic diagram showing a representative covalently linked tetravalent diabody with four epitope binding sites composed of two pairs of polypeptide chains (ie, four polypeptide chains total). . One polypeptide of each pair has CH2 and CH3 domains such that the associated chains form all or part of an Fc region. VL and VH domains that recognize the same epitope are indicated using the same shading or fill pattern. Two pairs of polypeptide chains may be the same. In embodiments where the two pairs of polypeptide chains are the same and the VL and VH domains recognize different epitopes (as shown in FIGS. 3A-3B), the resulting molecule has four binding sites, each binding bispecific and bivalent with respect to the epitope identified. In embodiments in which the VL and VH domains recognize the same epitope (eg, the same VL domain CDRs and the same VH domain CDRs are used for both chains), the resulting molecule has four epitope binding sites and a single monospecific and tetravalent for the epitope of Alternatively, the two pairs of polypeptides may be different. In embodiments in which the two pairs of polypeptide chains are different and the VL and VH domains of each pair of polypeptides recognize different epitopes (as indicated by the different shades and patterns in FIG. 3C), the resulting molecule has four epitopes. It has a binding site and is tetraspecific and monovalent for each bound epitope. FIG. 3A shows an Fc Region-containing diabodies comprising peptide heterodimer-promoting domains containing cysteine residues. FIG. 3B shows an Fc region-containing diabodies, which contain E-coil and K-coil heterodimer-promoting domains with cysteine residues and linkers (with optional cysteine residues). Figure 3C shows an Fc region-containing diabodies, which comprise antibody CH1 and CL domains. Figures 4A-4B provide schematic representations of representative covalent diabody molecules with two epitope binding sites composed of three polypeptide chains. Two of the polypeptide chains have CH2 and CH3 domains such that the attached chains form all or part of the Fc region. Polypeptide chains containing the VL and VH domains also contain a heterodimer-promoting domain. VL and VH domains that recognize the same epitope are indicated using the same shading or fill pattern. FIG. 5 provides a schematic representation of a representative covalently linked diabody molecule with four epitope binding sites composed of five polypeptide chains. Two of the polypeptide chains have CH2 and CH3 domains such that the joined chains form an Fc region that includes all or part of an Fc region. A polypeptide chain comprising linked VL and VH domains further comprises a heterodimer-promoting domain. VL and VH domains that recognize the same epitope are indicated using the same shading or fill pattern. Figures 6A-6F provide schematic representations of representative Fc region-containing trivalent binding molecules with three epitope binding sites. Figures 6A and 6B are trivalent binding molecules comprising two diabody-type binding domains and a Fab-type binding domain with different domain orientations, where the diabody-type binding domains are N-terminal or C-terminal to the Fc region, respectively. shows schematically the domains of . The molecules in Figures 6A and 6B contain four strands. Figures 6C and 6D depict two diabody-type binding domains N-terminal to the Fc region and a Fab-type binding domain in which the light and heavy chains are linked via a polypeptide spacer, or scFv-type binding, respectively. Schematically depicts the domains of a trivalent binding molecule, including domains. The trivalent binding molecules in FIGS. 6E and 6F each have two diabody-type binding domains C-terminal to the Fc region and a Fab-type binding in which the light and heavy chains are linked via a polypeptide spacer. Schematic representation of domains, or domains of trivalent binding molecules, including scFv-type binding domains. The trivalent binding molecules in Figures 6C-6F contain three strands. VL and VH domains that recognize the same epitope are indicated using the same shading or fill pattern. Figures 7A-7C present the results of an immunohistochemistry (IHC) study showing that MAB-A was induced by various non-small cell lung cancer types (Figure 7A, panels 1-8), breast cancer cells, prostate cancer cells, gastric cancer cells. ( FIG. 7B , panels 1-6), and colon cancer cells ( FIG. 7C , panels 1-8), while the isotype control was capable of specifically labeling any of these cancer cell types. It could not be specifically labeled (Figures 7A-7C). Figures 8A-8B present the results of cell staining studies showing that MAB-A binds human ADAM9 transiently expressed on the surface of 293-FT and CHO-K cells and to a lesser extent cynomolgus monkeys. Binding to ADAM9 (FIGS. 8A and 8B, respectively). Figures 9A-9B show the amino acid sequences of murine anti-ADAM9-VH domains (Figure 9A, SEQ ID NOs: 7, 16, 17, 18, 19, 21) aligned with several humanized/optimized variants of MAB-A. , 22, 23 and 28) and a murine anti-ADAM9-VL domain (FIG. 9B, SEQ ID NOS: 11, 51, 52, 53 and 54) aligned with several humanized/optimized variants of MAB-A. indicate. Positions that were substituted within the CDRs during the initial optimization are underlined as follows: potential deamidation and isomerization sites are single-underlined, lysine residues are double-underlined; Line underlining and additional labile residues are indicated by double-dashed underlining. Figures 10A-10B present ELISA binding curves of 10 selected optimized hMAB-A clones, including the CDR _H3 variant, parental hMAB-A (2.2), and an isotype control antibody. FIG. 10A presents the binding curve of cynoADAM9 and FIG. 10B presents the binding curve of huADAM9.

The present invention provides monospecific and bispecific, trispecific antibodies, including antibodies capable of specifically binding diabodies, BiTEs, and "disintegrin and metalloproteinase domain-containing protein 9"("ADAM9"). or molecules such as multispecific binding molecules. The invention particularly relates to binding molecules capable of exhibiting high affinity binding to human and non-human ADAM9. The invention more particularly relates to molecules thereby cross-reactive with human ADAM9 and non-human primate (eg, cynomolgus monkey) ADAM9. The invention further provides light chain variable (VL) domains and/or humanized and/or deimmunized to exhibit reduced immunogenicity upon administration of such ADAM9 binding molecules to a recipient subject. or any molecule containing a heavy chain variable (VH) domain. The invention also relates to pharmaceutical compositions comprising any such ADAM9 binding molecules, methods comprising the use of any such ADAM9 binding molecules in the treatment of cancer and other diseases and conditions.
I. Antibodies and their binding domains

Antibodies of the present invention are immunoglobulin molecules located in the variable domains of the immunoglobulin molecule capable of specific binding via at least one antigen recognition site to a target such as a carbohydrate, polynucleotide, lipid, or polypeptide. is. As used herein, the term "antibody and antibodies" includes monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, synthetic antibodies, chimeric antibodies, polyclonal antibodies, camelized Refers to antibodies, single-chain Fvs (scFv), single-chain antibodies, Fab fragments, F(ab') fragments, disulfide-linked bispecific Fvs (sdFv), intrabodies, and epitope-binding fragments of any of the foregoing . In particular, the term "antibody" includes immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, ie, molecules that contain an epitope binding site. Immunoglobulin molecules are of any type ₍ e.g. IgG _, IgE, IgM, IgD, IgA and IgY), _{class (} e.g. IgG1, IgG2, IgG3, IgG4, _IgA1 and _IgA2 ) or subclass can be In recent decades, there has been a resurgence of interest in therapeutically promising antibodies, which have become one of the top biotechnology-derived drugs (Chan, CE et al. (2009)). “The Use Of Antibodies In The Treatment Of Infectious Diseases,” Singapore Med. J. 50(7):663-666). In addition to their use in diagnostics, antibodies have proven useful as therapeutic agents. Over 200 antibody-based drugs have been approved for use or are in development.

An antibody is capable of "immunospecifically binding" to a polypeptide or protein or non-protein molecule because of the presence of specific domains or portions or conformations ("epitopes") on such molecule. . Epitope-containing molecules can have immunogenic activity so as to elicit an antibody-producing response in an animal; such molecules are referred to as "antigens." As used herein, an antibody, diabody, or other epitope-binding molecule binds to a region of another molecule (i.e., an epitope) more frequently and more rapidly than to another epitope. It is said to bind "immunospecifically" if it reacts or associates with a substance for a longer period of time and/or with a higher affinity. For example, an antibody that immunospecifically binds to a viral epitope will more readily, with higher affinity, avidity, than it will immunospecifically bind to other viral or non-viral epitopes. and/or antibodies that bind epitopes of the virus for longer periods of time. By reading this definition, for example, an antibody (or portion or epitope) that immunospecifically binds to a first target may or may not specifically or preferentially bind to a second target. It is also understood that Thus, "immunospecific binding" to a particular epitope does not necessarily require (although it can include) exclusive binding to that epitope. Generally, but not necessarily, references to binding mean "immunospecific" binding. Two molecules are said to bind to each other in a "physiospecific" manner if such binding exhibits the specificity with which the receptors bind their respective ligands.

The term "monoclonal antibody" refers to a homogenous antibody population composed of the amino acids (naturally occurring or non-naturally occurring) that the monoclonal antibody participates in selectively binding an antigen. Monoclonal antibodies are highly specific, being directed against a single epitope (or antigenic site). The term "monoclonal antibody" includes not only intact and full-length monoclonal antibodies, but also fragments thereof (e.g., Fab, Fab', F(ab') _2Fv ), single chain (scFv), fragment thereof Mutants, fusion proteins containing antibody portions, humanized monoclonal antibodies, chimeric monoclonal antibodies, and any other modified configuration of the immunoglobulin molecule that contains an antigen recognition site of the required specificity and the ability to bind antigen. contain. The term is not intended to be limited as to the source of the antibody or the manner in which it is produced (eg, by hybridoma, phage selection, recombinant expression, transgenic animals, etc.). The term includes whole immunoglobulins as well as fragments and the like described above under the definition of "antibody." Methods for making monoclonal antibodies are known in the art. One method that can be employed is that of Kohler, G. et al. (1975) "Continuous Cultures Of Fused Cells Secreting Antibody Of Predefined Specificity," Nature 256:495-497, or variations thereof. Monoclonal antibodies are typically raised in mice, rats or rabbits. Antibodies are produced by immunizing an animal with an immunogenic amount of cells, cell extracts, or protein preparations containing the desired epitope. Immunogens can be, but are not limited to, primary cells, cultured cell lines, cancerous cells, proteins, peptides, nucleic acids, or tissues. Cells used for immunization can be cultured for a period of time (eg, at least 24 hours) prior to their use as immunogens. Cells can be used as immunogens alone or in combination with non-denaturing adjuvants such as Ribi (see, e.g., Jennings, VM (1995) "Review of Selected Adjuvants Used in Antibody Production," ILAR J. 37(3)). ):119-125). In general, cells should be kept intact and preferably viable when used as immunogens. Intact cells can allow antigens to be better detected by immunized animals than burst cells. The use of denaturing or harsh adjuvants, such as Freud's adjuvant, may rupture the cells and is therefore not recommended. The immunogen can be administered multiple times, at regular intervals, such as twice weekly, or weekly, or administered in such a way as to maintain viability in the animal (e.g., in tissue recombinants). can do. Alternatively, existing monoclonal antibodies and any other equivalent antibodies immunospecific for the desired pathogenic epitope can be sequenced and recombinantly produced by any means known in the art. can do. In one embodiment, such antibodies are sequenced and the polynucleotide sequences are then cloned into vectors for expression or propagation. The sequences encoding the antibody of interest can be maintained in the vector in the host cell, which can then be expanded and frozen for future use. The polynucleotide sequences of such antibodies are used for genetic engineering to generate monospecific or multispecific (eg, bispecific, trispecific and tetraspecific) molecules and affinity antibodies of the invention. Optimized, chimeric, humanized, and/or caninized antibodies can be generated to improve affinity or other properties of antibodies. A general principle in humanizing an antibody involves retaining the basic sequence of the antigen-binding portion of the antibody, while replacing the non-human remainder of the antibody with human antibody sequences.

A natural antibody (eg, a natural IgG antibody) is composed of two "light chains" complexed with two "heavy chains". Each light chain comprises a variable domain (“VL”) and a constant domain (“CL”). Each heavy chain comprises a variable domain (“VH”), three constant domains (“CH1”, “CH2” and “CH3”) and a “hinge” region (“H”) located between the CH1 and CH2 domains. including. The basic structural unit of naturally occurring immunoglobulins (eg, IgG) is thus a tetramer with two light chains and two heavy chains, commonly represented as a glycoprotein of about 150,000 Da. The amino-terminal (“N-terminal”) portion of each chain includes a variable domain of about 100-110 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal (“C-terminal”) portion of each chain defines a constant region, with light chains having a single constant domain and heavy chains usually having three constant domains plus a hinge region. Thus, the structure of the light chain of an IgG molecule is n-VL-CL-c and the structure of the IgG heavy chain is n-VH-CH1-H-CH2-CH3-c, where n and c are represent the N-terminus and C-terminus of the polypeptide). The variable domain of an IgG molecule consists of one, two, and most commonly three complementarity determining regions (“CDRs”, ie, CDR1, CDR2 and CDR3, respectively), which are the residues that contact the epitope. groups and non-CDR segments, termed framework regions (“FRs”), which generally maintain structure and may make such contacts (although certain framework residues may also contact epitopes). Position the CDR regions as possible. Thus, the VL and VH domains typically have the structure: n-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-c, where "n" refers to the N-terminus and "c" refers to the C end). Polypeptides that are the first, second, third, and fourth FRs of an antibody light chain (or that can function as the first, second, third, and fourth FRs of an antibody light chain) are described herein designated therein as: FR _L 1 domain, FR _L 2 domain, FR _L 3 domain, and FR _L 4 domain. Similarly, polypeptides that are the first, second, third and fourth FRs of a heavy chain of an antibody (or that may function as the first, second, third and fourth FRs of a heavy chain of an antibody) are Each is designated herein as: FR _H 1 domain, FR _H 2 domain, FR _H 3 domain and FR _H 4 domain. Polypeptides that are the first, second and third CDRs of a light chain of an antibody (or that can function as the first, second and third CDRs of a light chain of an antibody) are referred to herein as: CDR _L 1 designated domains, CDR _L 2 domain, and CDR _L 3 domain. Similarly, polypeptides that are the first, second and third CDRs of an antibody heavy chain (or that may function as the first, second and third CDRs of an antibody heavy chain) are referred to herein as: CDR _H designated as CDR H 1 domain, CDR _H 2 domain, and CDR _H 3 domain. Thus, the terms CDR _L 1 domain, CDR _L 2 domain, CDR _L 3 domain, CDR _H 1 domain, CDR _H 2 domain, and CDR _H 3 domain refer to proteins such as Whether the protein is an antibody having light and heavy chains, or a diabody or single chain binding molecule (e.g., scFv, BiTe, etc.), or another type of protein, Of interest are polypeptides that are capable of binding to an epitope.

Thus, as used herein, the term "epitope-binding fragment" refers to a fragment of an antibody capable of immunospecifically binding to an epitope, and the term "epitope-binding site" refers to the epitope-binding Refers to the part of the molecule that contains the fragment. An epitope-binding fragment may comprise any one, two, three, four, or five CDR domains of an antibody, or may comprise all six of the CDR domains of an antibody and is immunospecific for such an epitope. It is capable of binding specifically, but may exhibit immunospecificity, affinity or selectivity for an epitope different from that of such antibodies. Preferably, however, the epitope-binding fragment includes all six of the CDR domains of such antibodies. An epitope-binding fragment of an antibody can be a single polypeptide chain (eg, scFv) or can comprise two or more polypeptide chains, each having an amino and carboxy terminus (eg, a body, Fab fragment, Fab ₂ fragment, etc.). Unless otherwise stated, the order of the domains of the protein molecules described herein is in the "N-terminal to C-terminal" direction.

The invention specifically provides single chain variable domain fragments (“scFv”) comprising the anti-ADAM9-VL and/or VH domains of the invention and multispecific binding molecules comprising such anti-ADAM9-VL and/or VH domains. contain. Single-chain variable domain fragments comprise the VL and VH domains linked together using a short "linker" peptide. Such linkers can be modified to provide additional functions, such as, for example, permitting attachment of drugs or permitting attachment of solid supports. Single-chain variants can be produced either recombinantly or synthetically. An automated synthesizer can be used for synthetic production of scFv. For recombinant production of scFv, a suitable plasmid containing a polynucleotide encoding the scFv is transferred to a suitable host cell, either eukaryotic such as yeast, plant, insect or mammalian cells, or prokaryotic such as E. coli. can be introduced into Polynucleotides encoding the scFv of interest can be made by routine manipulations such as ligation of polynucleotides. The resulting scFv can be isolated using standard protein purification techniques known in the art.

The invention also provides the CDR _H 1, CDR _H 2, CDR _H 3, CDR _L 1, CDR _L 2, and CDR _L 3 domains of the humanized variants of the anti-ADAM9 antibodies of the invention, as well as the CDR _L Also specifically included are VL domains comprising any 1, 2 or 3 and _VH domains comprising any 1, 2 or 3 of such CDR Hs and multispecific binding molecules comprising the same. The term "humanized" antibody refers to chimeric molecules that have the epitope binding sites of an immunoglobulin from a non-human species and residual immunoglobulin structures based on human immunoglobulin structures and/or sequences. Humanized antibodies are generally prepared using recombinant techniques. Anti-ADAM9 antibodies of the invention include humanized, chimeric or canine variants of the antibody designated herein as "MAB-A". Polynucleotide sequences encoding variable domains of MAB-A are used for genetic engineering to produce MAB-A derivatives with improved or altered characteristics (eg, affinity, cross-reactivity, specificity, etc.). can be generated. A general principle in humanizing an antibody involves retaining the basic sequence of the epitope-binding portion of the antibody while replacing the non-human remainder of the antibody with human antibody sequences. There are four general steps for humanizing monoclonal antibodies. These are: (1) determining the nucleotide and predicted amino acid sequences of the starting antibody light and heavy chain variable domains; (2) designing a humanized or caninized antibody, i.e. during the humanization or caninization process (3) adopting the actual humanization or caninization methods/techniques; and (4) transfecting and expressing the humanized antibody. See, for example, U.S. Patent Nos. 4,816,567; 5,807,715; 5,866,692; and 6,331,415. The term "optimized" antibody refers to a variable light or heavy chain that confers higher binding affinity, e.g., 2-fold or more, higher binding affinity to human ADAM9 and/or cynomolgus monkey ADAM9 compared to the parent antibody. Refers to an antibody that has at least one amino acid that differs from the parent antibody in at least one complementarity determining region (CDR) in a region. It will be appreciated from the teachings presented herein that the antibodies of the invention may be humanized, optimized, or both humanized and optimized.

Epitope binding sites may comprise either complete variable domains fused to one or more constant domains, or only the CDRs of such variable domains grafted onto suitable framework regions. Epitope binding sites may be wild-type or modified by one or more amino acid substitutions, insertions or deletions. Such action partially or completely precludes the ability of constant regions to function as immunogens in recipients (e.g., human individuals), but leaves the possibility of an immune response to foreign variable domains (LoBuglio, A.F. et al. (1989) "Mouse/Human Chimeric Monoclonal Antibody In Man: Kinetics And Immune Response," Proc. Natl. Acad. Sci. (U.S.A.) 86:4220-4224). Another approach focuses not only on providing constant regions of human origin, but also on modifying the variable domains to reshape them as closely as possible to the form found in human immunoglobulins. The variable domains of both the light and heavy chains of antibodies contain three CDRs, flanked by four framework regions, that change in response to the antigen of interest and determine binding ability, which are associated with a given species. It is known to be relatively conserved in and putatively provide scaffolding for the CDRs. When a non-human antibody is prepared for a specific antigen, the variable domain is "reshaped" or "humanized" by grafting CDRs derived from the non-human antibody onto the FRs present in the modified human antibody. can do. Application of this approach to a variety of antibodies is described in Sato, K. et al. (1993) Cancer Res 53:851-856. Riechmann, L. et al. (1988) "Reshaping Human Antibodies for Therapy," Nature 332. Verhoeyen, M. et al. (1988) "Reshaping Human Antibodies: Grafting An Antilysozyme Activity," Science 239:1534-1536; Kettleborough, C. A. et al. (1991) "Humanization Of A Mouse Monoclonal Antibody By CDR-Grafting: The Importance Of Framework Residues On Loop Conformation,” Protein Engineering 4:773-3783; Maeda, H. et al. (1991) “Construction Of Reshaped Human Antibodies With HIV-Neutralizing Activity,” Human Antibodies Hybridoma 2: 124-134; Gorman, S. D. et al. (1991) "Reshaping A Therapeutic CD4 Antibody," Proc. Natl. Acad. Sci. (U.S.A.) 88:4181-4185; Human Monoclonal Antibody To Inhibit Human Respiratory Syncytial Virus Infection in vivo,” Bio/Technology 9:266-271; Co, M. S. et al. (1991) “Humanized Antibodies For Antiviral Therapy,” Proc. Natl. Acad. ) 88:2869-2873; Carter, P. et al. (1992) "Humanization Of An Anti-p185her2 Antibody For Human Cancer Therapy," Proc. Natl. Acad. Sci. (U.S.A.) 89:4285-4289; Humanized Antibodies With Specificity For The CD33 Antigen,” J. Immunol. 148:1149-1154. In some embodiments, a humanized antibody preserves all CDR sequences (eg, a humanized murine antibody that includes all six of the CDRs present in the murine antibody). In other embodiments, the humanized antibody has one or more CDRs (1, 2, 3, 4, 5, or 6) that differ in sequence relative to the CDRs of the original antibody.

Numerous human containing epitope binding sites derived from non-human immunoglobulins, including chimeric antibodies having rodent or modified rodent variable domains and their associated complementarity determining regions (CDRs) fused to human constant domains. (1991) "Man-made Antibodies," Nature 349:293-299; Lobuglio et al. (1989) "Mouse/Human Chimeric Monoclonal Antibody In Man: Kinetics Sci. (USA) 86:4220-4224; Shaw et al. (1987) “Characterization Of A Mouse/Human Chimeric Monoclonal Antibody (17-1A) To A Colon Cancer Tumor- Associated Antigen," J. Immunol. 138:4534-4538; and Brown et al. (1987) "Tumor-Specific Genetically Engineered Murine/Human Chimeric Monoclonal Antibody," Cancer Res. 47:3577-3583). Others have described rodent CDRs grafted onto human bearing framework regions (FRs) prior to fusion with appropriate human antibody constant domains (e.g. Riechmann, L. et al. (1988) "Reshaping Human Antibodies for Therapy," Nature 332:323-327; Verhoeyen, M. et al. (1988) "Reshaping Human Antibodies: Grafting An Antilysozyme Activity," Science 239:1534-1536; ) “Replacing The Complementarity-Determining Regions In A Human Antibody With Those From A Mouse,” Nature 321:522-525). Another document describes rodent CDRs carried by recombinantly veneered rodent framework regions (see, eg, European Patent Publication No. 519,596). These "humanized" molecules are designed to minimize unwanted immune responses to rodent anti-human antibody molecules, which limits the duration and efficacy of their therapeutic application in human recipients. . Other methods of humanizing antibodies that are also available are Daugherty et al. (1991) "Polymerase Chain Reaction Facilitates The Cloning, CDR-Grafting, And Rapid Expression Of A Murine Monoclonal Antibody Directed Against The CD18 Component Of Leukocyte Integrins," Nucl. Acids Res. 19:2471-2476 and U.S. Patent Nos. 6,180,377; 6,054,297; 5,997,867; ing.
II. Fcγ receptor (FcγR)

The CH2 and CH3 domains of the two heavy chains of an antibody interact to form the "Fc region", which includes, but is not limited to, Fcγ receptors ("FcγR"), cellular " It is the domain recognized by the 'Fc receptor'. As used herein, "Fc region" is used to define the C-terminal region of an IgG heavy chain including the CH2 and CH3 domains of that chain. An Fc region is of a particular IgG isotype, class or subclass if its amino acid sequence is most homologous to that IgG isotype, class or subclass compared to other IgG isotypes. be done.

The amino acid sequence of an exemplary human IgG1 CH2-CH3 domain numbered with the EU index as described in Kabat (SEQ ID NO: 1):
231 240 250 260 270 280
APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD
290 300 310 320 330
GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA
340 350 360 370 380
PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE
390 400 410 420 430
WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE
440 447
ALHNHYTQKS LSLSPG X
where X is lysine (K) or absent.

The amino acid sequence of an exemplary human IgG2 CH2-CH3 domain, numbered by the EU index as described in Kabat, (SEQ ID NO: 2):
231 240 250 260 270 280
APPVA-GPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVQFNWYVD
290 300 310 320 330
GVEVHNAKTK PREEQFNSTF RVVSVLTVVH QDWLNGKEYK CKVSNKGLPA
340 350 360 370 380
PIEKTISKTK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDISVE
390 400 410 420 430
WESNGQPENN YKTTPPMLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE
440 447
ALHNHYTQKS LSLSPG X
where X is lysine (K) or absent.

The amino acid sequence of an exemplary human IgG3 CH2-CH3 domain numbered with the EU index as described in Kabat (SEQ ID NO:3):
231 240 250 260 270 280
APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVQFKWYVD
290 300 310 320 330
GVEVHNAKTK PREEQYNSTF RVVSVLTVLH QDWLNGKEYK CKVSNKALPA
340 350 360 370 380
PIEKTISKTK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE
390 400 410 420 430
WESSGQPENN YNTTPPMLDS DGSFFLYSKL TVDKSRWQQG NIFSCSVMHE
440 447
ALHNRFTQKSLSLSPGX
where X is lysine (K) or absent.

The amino acid sequence of an exemplary human IgG4 CH2-CH3 domain numbered with the EU index as described in Kabat (SEQ ID NO: 4):
231 240 250 260 270 280
APEFLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSQED PEVQFNWYVD
290 300 310 320 330
GVEVHNAKTK PREEQFNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKGLPS
340 350 360 370 380
SIEKTISKAK GQPREPQVYT LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE
390 400 410 420 430
WESNGQPENN YKTTPPVLDS DGSFFLYSRL TVDKSRWQEG NVFSCSVMHE
440 447
ALHNHYTQKS LSLSLG X
where X is lysine (K) or absent.

Throughout the specification, residue numbering in the constant region of IgG heavy chains is hereby expressly incorporated by reference, Kabat et al., Sequences of Proteins of Immunological Interest, 5th ^Ed . Public Health Service , NH1, MD (1991) ("Kabat") is the numbering of the EU index. The term "EU index as described in Kabat" refers to the constant domain numbering of the human IgG1 EU antibody provided in Kabat. Amino acids from the variable domains of the mature heavy and light chains of immunoglobulins are designated by the position of the amino acid in the chain. Kabat describes many amino acid sequences of antibodies, identifies amino acid consensus sequences for each subgroup, assigns residue numbers to each amino acid, and CDRs are identified as defined by Kabat (Chothia , C. & Lesk, AM ((1987) "Canonical structures for the hypervariable regions of immunoglobulins," J. Mol. Biol. 196: _901-917 ). understood to start). Kabat's numbering scheme is extendable to antibodies not included in his list by aligning the antibody in question with one of the consensus sequences in Kabat by referencing the conserved amino acids. This method for assigning residue numbers has become standard in the art and readily identifies amino acids at equivalent positions in different antibodies, including chimeric or humanized variants. For example, the amino acid at position 50 of a human antibody light chain occupies the equivalent position to the amino acid at position 50 of a mouse antibody light chain.

Polymorphisms are observed at a number of different positions within the antibody constant region (e.g., but not limited to positions 270, 272, 312, 315, numbered by the EU index as described in Kabat, Fc positions including 356, and 358), so there may be some differences between the sequences presented and those in the prior art. Polymorphs of human immunoglobulins have been well characterized. Currently, 18 Gm allotypes are known: G1m(1,2,3,17) or G1m(a,x,f,z), G2m(23) or G2m(n), G3m(5,6, 10, 11, 13, 14, 15, 16, 21, 24, 26, 27, 28) or G3m (b1, c3, b3, b0, b3, b4, s, t, g1, c5, u, v, g5 ) (Lefranc, et al., "The Human IgG Subclasses: Molecular Analysis of Structure, Function And Regulation." Pergamon, Oxford, pp. 43-78 (1990); Lefranc, G. et al., 1979, Hum. Genet .: 50, 199-211). It is specifically contemplated that the antibodies of the present invention may incorporate any allotype, isoallotype, or haplotype of any immunoglobulin gene, and are not limited to allotypes, isoallotypes, or haplotypes of the sequences provided herein. be done. Furthermore, in some expression systems, the C-terminal amino acid residues of the CH3 domain (in bold above) can be removed post-translationally. Accordingly, the C-terminal residue of the CH3 domain is any amino acid residue in the ADAM9-binding molecules of the invention. Specifically encompassed by the present invention are ADAM9 binding molecules that lack the C-terminal residues of the CH3 domain. Such constructs containing the C-terminal lysine residue of the CH3 domain are also specifically included in the present invention.

As mentioned above, the Fc region of natural IgG antibodies is capable of binding to cellular Fcγ receptors (FcγR). Such binding results in transduction of activating or inhibitory signals to the immune system. The ability of such binding to result in diametrically opposed functions reflects structural differences between different FcγRs, particularly those that bind to immunoreceptor tyrosine-based activation motifs (“ITAMs”) or immunoreceptor tyrosine-based activation motifs (“ITAMs”). Reflects whether or not it has an inhibition motif (“ITIM”). The recruitment of different cytoplasmic enzymes to these structures determines the outcome of FcγR-mediated cellular responses. ITAM-containing FcγRs include FcγRI, FcγRIIA, FcγRIIIA, and activate immunization when bound to Fc regions (eg, aggregated Fc regions present in immune complexes). FcγRIIB is the only currently known natural ITIM-containing FcγR; it acts to suppress or inhibit the immune system when bound to aggregated Fc regions. Human neutrophils express the FcγRIIA gene. FcγRIIA clustering or specific antibody cross-linking via immune complexes serves to aggregate ITAMs with receptor-associated kinases that promote ITAM phosphorylation, which serves as a docking site for Syk kinase, Its activation results in the activation of downstream substrates (eg PI ₃ K). Cellular activation leads to the release of proinflammatory mediators. The FcγRIIB gene is expressed on B lymphocytes; its extracellular domain is 96% identical to FcγRIIA and binds IgG complexes in an indistinguishable manner. The presence of ITIMs in the cytoplasmic domain of FcγRIIB defines this inhibitory subclass of FcγRs. Recently, the molecular basis for this inhibition has been established. When co-ligated with activating FcγRs, ITIMs in FcγRIIB become phosphorylated and inositol polyphosphate 5′ hydrolyzes phosphoinositol messengers released as a result of ITAM-containing FcγR-mediated tyrosine kinase activation. - Attracts the SH2 domain of the phosphatase (SHIP) and consequently prevents the influx of intracellular Ca ⁺⁺ . Thus, cross-linking of FcγRIIB suppresses the activating response to FcγR ligation and inhibits cellular responsiveness. B-cell activation, B-cell proliferation and antibody secretion are thus abolished.
III. Bispecific Antibodies, Multispecific Diabodies and DART® Diabodies

The ability of an antibody to bind an epitope of an antigen depends on the presence and amino acid sequence of the VL and VH domains of the antibody. The interaction of the light and heavy chains of an antibody, and particularly the interaction of its VL and VH domains, form one of the two epitope binding sites of natural antibodies such as IgG. Although natural antibodies can only bind one epitope species (i.e. they are monospecific), they can bind multiple copies of that epitope species (i.e. bivalent or showing multivalency).

Antibody functionality is enhanced by generating multispecific antibody-based molecules that can simultaneously bind two separate and independent antigens (or different epitopes of the same antigen) and/or against the same epitope and/or antigen. Enhancement can be achieved by generating antibody-based molecules with higher valencies (ie, more than two binding sites).

Various recombinant bispecific antibody formats have been developed to provide molecules with higher potency than natural antibodies (e.g. PCT Publication Nos. WO2008/003116, WO2009/132876, see WO2008/003103, WO2007/146968, WO2009/018386, WO2012/009544, WO2013/070565), most of which are further epitope binding fragments. (e.g. scFv, VL, VH, etc.) to or within an antibody core (IgA, IgD, IgE, IgG or IgM) or multiple epitope binding fragments (e.g. two Fab fragments or scFv) A linker peptide is used to fuse the Another format uses a linker peptide to fuse an epitope-binding fragment (eg, scFv, VL, VH, etc.) to a dimerization domain such as a CH2-CH3 domain or another polypeptide (eg, PCT Publication No. International See Publication No. 2005/070966, WO2006/107786A, WO2006/107617A, WO2007/046893). PCT Publication Nos. WO 2013/174873, WO 2011/133886 and WO 2010/136172 disclose that the CL and CH1 domains are switched from their respective natural positions and the VL and VH domains are diversified. (see, e.g., PCT Publication No. WO 2008/027236; WO 2010/108127), disclosing trispecific antibodies that are capable of binding to more than one antigen. is doing. PCT Publication Nos. WO2013/163427 and WO2013/119903 disclose modifying the CH2 domain to include a fusion protein adduct comprising a binding domain. PCT Publication Nos. WO 2010/028797, WO 2010/028796 and WO 2010/028795 replace the Fc region with additional VL and VH domains to form trivalent binding molecules Disclosed are recombinant antibodies. PCT Publication Nos. WO 2003/025018 and WO 2003/012069 disclose recombinant diabodies, each chain of which comprises an scFv domain. PCT Publication No. WO 2013/006544 discloses multivalent Fab molecules synthesized as single polypeptide chains and then proteolyzed to form heterodimeric structures. PCT Publication No. WO 2014/022540, WO 2013/003652, WO 2012/162583, WO 2012/156430, WO 2011/086091, WO 2008/024188 WO 2007/024715, WO 2007/075270, WO 1998/002463, WO 1992/022583 and WO 1991/003493 disclose additional binding domains or functional groups in antibodies or adding to an antibody portion (e.g., adding a diabody to the antibody light chain, or adding additional VL and VH domains to the antibody light and heavy chain, or adding a heterologous fusion protein, or linking multiple Fab domains together in a chain).

Diabody design is based on antibody derivatives known as single-chain variable domain fragments (scFv). Such molecules are made by linking light and/or heavy chain variable domains using short linking peptides. Bird, R. E. et al. (1988) ("Single-Chain Antigen-Binding Proteins," Science 242:423-426) describe a linkage that bridges approximately 3.5 nm between the carboxy terminus of one variable domain and the amino terminus of another. Examples of peptides are given. Linkers of other sequences have been designed and used (Bird et al. (1988) "Single-Chain Antigen-Binding Proteins," Science 242:423-426). The linker can then be modified for additional functions such as attachment of drugs or attachment to solid supports. Single-chain variants can be produced either recombinantly or synthetically. For synthetic production of scFv, an automated synthesizer can be used. For recombinant production of scFv, suitable plasmids containing the scFv-encoding polynucleotide are added to either eukaryotes such as yeast, plant, insect or mammalian cells, or prokaryotes such as E. coli. can be introduced into a suitable host cell of Polynucleotides encoding the scFv of interest can be made by routine manipulations such as ligation of polynucleotides. The resulting scFv can be isolated using standard protein purification techniques known in the art.

Those skilled in the art differ from such natural antibodies in that they are capable of binding two or more different epitope species (i.e. exhibit bispecific or multispecificity in addition to being bivalent or multivalent). It has been shown that diabodies can be produced (see, for example, Holliger, P. et al. (1993) "'Diabodies': Small Bivalent And Bispecific Antibody Fragments," Proc. Natl. Acad. Sci. (U.S.A.) 90:6444- US 2004/0058400 (Hollinger et al.); US 2004/0220388 / WO 02/02781 (Mertens et al.); Alt et al. (1999) FEBS Lett. 454(1-2):90-94; Lu, D. et al. (2005) “A Fully Human Recombinant IgG-Like Bispecific Antibody To Both The Epidermal Growth Factor Receptor And The Insulin-Like Growth Factor Receptor For Enhanced Antitumor Activity,” J. Biol. Chem. ): 19665-19672; WO 02/02781 (Mertens et al.); Olafsen, T. et al. (2004) "Covalent Disulfide-Linked Anti-CEA Diabody Allows Site-Specific Conjugation And Radiolabeling For Tumor Targeting Applications," Protein Eng. Des. Sel. 17(1):21-27; Wu, A. et al. (2001) “Multimerization Of A Chimeric Anti-CD20 Single Chain Fv-Fv Fusion Protein Is Mediated Through Variable Domain Exchange,” Protein Engineering 14(2): 1025-1033; Asano et al. (2004) "A Diabody For Cancer Immunotherapy And Its Functional Enhancement By Fusion Of Human Fc Domain," Abstract 3P-683, J. Biochem. 76(8):992; Takemura, S. et al. al. (2000) "Construction Of A Diabody (Small Recombinant Bispecific Antibody) Using A Refolding System," Protein Eng. 13(8):583-588; Baeuerle, P.A. et al. (2009) "Bispecific T-Cell Engaging Antibodies For Cancer Therapy," Cancer Res. 69(12):4941-4944).

Provision of bispecific binding molecules (e.g., non-monospecific diabodies) is sufficient, but not limited, to co-ligate and/or co-localize different cells expressing different epitopes. significant advantages over antibodies, including sufficient "trans" binding capacity and/or sufficient "cis" binding capacity to co-ligate and/or co-localize different molecules expressed by the same cell. I will provide a. Bispecific binding molecules (eg, non-monospecific diabodies) therefore find a wide variety of applications, including therapy and immunodiagnostics. Bispecificity allows great flexibility in the design and engineering of diabodies in a variety of applications, including enhanced avidity to multimeric antigens, cross-linking of different antigens, and specific binding properties dependent on the presence of both target antigens. Provides directed targeting to cell types. Due to their increased valence, low dissociation rate and rapid clearance from circulation (for small size diabodies of about 50 kDa or less), diabody molecules known in the art are also useful in the field of tumor imaging. (Fitzgerald et al. (1997) "Improved Tumor Targeting By Disulphide Stabilized Diabodies Expressed In Pichia pastoris," Protein Eng. 10:1221-1225).

The ability to produce bispecific diabodies can be achieved by co-ligating two cells together (e.g., cytotoxic T cells and tumor cells) by co-ligating receptors present on the surface of different cells. (Staerz et al. (1985) "Hybrid Antibodies Can Target Sites For Attack By T Cells," Nature 314:628-631; Holliger et al. (1996). “Specific Killing Of Lymphoma Cells By Cytotoxic T-Cells Mediated By A Bispecific Diabody,” Protein Eng. 9:299-305; and Marvin et al. (2005) “Recombinant Approaches To IgG-Like Bispecific Antibodies,” Acta Pharmacol. 26:649-658). Alternatively (or additionally), bispecific (or trispecific or trispecific multispecific) diabodies to co-ligate molecules such as receptors present on the surface of the same cell. (in "cis") can be used. Co-ligation of different cells and/or receptors is useful for modulating effector function and/or immune cell signaling. Multispecific molecules (e.g., bispecific diabodies) containing epitope binding sites are CD2, CD3, expressed on T lymphocytes, natural killer (NK) cells, antigen presenting cells or other mononuclear cells. It can be directed to any immune cell surface determinant such as CD8, CD16, T cell receptor (TCR), NKG2D, and the like. In particular, epitope binding sites directed to cell surface receptors present on immune effector cells are useful in generating polyspecific binding molecules capable of mediating redirected cell killing.

However, said advantages come at a considerable cost. The formation of such non-monospecific diabodies requires the successful assembly of two or more distinct and distinct polypeptides (i.e., such formation is a heterodimer of different polypeptide chain species where the diabodies (needs to be formed through transformation). This fact is in contrast to monospecific diabodies formed by homodimerization of the same polypeptide chain. Since at least two different polypeptides (i.e., two polypeptide species) must be provided to form a non-monospecific diabodies, homodimerization of such polypeptides is also inactive. (Takemura, S. et al. (2000) “Construction Of A Diabody (Small Recombinant Bispecific Antibody) Using A Refolding System,” Protein Eng. 13(8):583-588). Production of peptides must be carried out in a manner that prevents covalent binding between polypeptides of the same species (i.e., to prevent homodimerization) (Takemura, S. et al. (2000) “Construction Of A Diabody (Small Recombinant Bispecific Antibody) Using A Refolding System, "Protein Eng. 13(8):583-588). The art teaches non-covalent association of such polypeptides (see, for example, Olafsen et al. (2004) “Covalent Disulfide-Linked Anti-CEA Diabody Allows Site-Specific Conjugation And Radiolabeling For Tumor Targeting Applications Des. Sel. 17:21-27; Asano et al. (2004) “A Diabody For Cancer Immunotherapy And Its Functional Enhancement By Fusion Of Human Fc Domain,” Abstract 3P-683, J. Biochem. 76(8):992; Takemura, S. et al. (2000) “Construction Of A Diabody (Small Recombinant Bispecific Antibody) Using A Refolding System,” Protein Eng. 13(8):583-588; et al. (2005) "A Fully Human Recombinant IgG-Like Bispecific Antibody To Both The Epidermal Growth Factor Receptor And The Insulin-Like Growth Factor Receptor For Enhanced Antitumor Activity," J. Biol. Chem. 280(20):19665 -19672).

However, those skilled in the art recognize that bispecific diabodies composed of non-covalently linked polypeptides are unstable and readily dissociate into non-functional monomers (e.g., Lu, D et al. (2005) "A Fully Human Recombinant IgG-Like Bispecific Antibody To Both The Epidermal Growth Factor Receptor And The Insulin-Like Growth Factor Receptor For Enhanced Antitumor Activity," J. Biol. Chem. 280(20):19665 -19672).

Faced with this challenge, those skilled in the art have successfully developed a stable, covalently linked, heterodimeric, non-monospecific diabody termed the DART® diabody; , 296,816 and 9,284,375 and U.S. Patent Publication Nos. 2015/0175697; 2014/0255407; 2014/0099318; 2013/0295121; WO 2012/162068; WO 2010/0174053; WO 2010/080538; 2009/0060910; 2007-0004909; EP 1868650; and PCT Publication Nos. WO 2012/162068; WO 2012/018687; WO 2010/080538; WO 2006/113665; Targeting HIV Reservoir in Infected CD4 T Cells by Dual-Affinity Re-targeting Molecules (DARTs) that Bind HIV Envelope and Recruit Cytotoxic T Cells,” PLoS Pathog. 11(11):e1005233. doi: 10.1371/journal.ppat.1005233; Al Hussaini, M. et al. (2015) “Targeting CD123 In AML Using A T-Cell Directed Dual-Affinity Re-Targeting (DART®) Platform,” Blood pii: blood-2014-05-575704; Chichili, G.R. et al. al. (2015) “A CD3xCD123 Bispecific DART For Redirecting Host T Cells To Myelogenous Leukemia: Preclinical Activity And Safety In Nonhuman Primates,” Sci. Transl. Med. 7(289):289ra82; Moore, P.A. et al. (2011) "Application Of Dual Affinity Retargeting Molecules To Achieve Optimal Redirected T-Cell Killing Of B-Cell Lymphoma," Blood 117(17): 4542-4551; Veri, M.C. et al. (2010) "Therapeutic Control Of B Cell Activation Via Recruitment Of Fcgamma Receptor IIb (CD32B) Inhibitory Function With A Novel Bispecific Antibody Scaffold," Arthritis Rheum. 62(7):1933-1943 and Johnson, S. et al. (2010) "Effector Cell Recruitment With Novel Fv-Based Dual-Affinity Re-Targeting Protein Leads To Potent Tumor Cytolysis And in vivo B-Cell Depletion," J. Mol. Biol. 399( 3):436-449). Such diabodies comprise two or more covalently complexed polypeptides, wherein one or more of the cysteine residues are engineered to allow disulfide bonds to form. and thereby covalently linking one or more pairs of such polypeptide chains to each other. For example, the addition of a cysteine residue to the C-terminus of such constructs allows for disulfide bonding between the polypeptide chains involved, allowing the resulting diabody to It has been shown to stabilize. Such molecules can be bispecific (or multispecific) and thus can co-ligate two or more molecules. Such co-ligation makes it possible to provide enhanced immunotherapy. Furthermore, because the individual polypeptide chains of such molecules form covalently bound complexes, the molecules exhibit much greater stability than diabodies containing non-covalently bound polypeptide chains.

Recently, trivalent and multivalent molecules incorporating two diabody-type binding domains and one non-diabody-type domain and an Fc region have been described (e.g., PCT Publication No. WO 2015/184207 and WO 2015/184207 and 2015/184203). Such binding molecules can be used to generate monospecific, bispecific or trispecific molecules. The ability to bind three different epitopes provides enhanced potency.

Another construct is a tetravalent tandem form, also called "TandAb", formed by homodimerization of two identical polypeptide chains, each having VH1, VL2, VH2, and VL2 domains, but is not limited to Known in the art for applications where tetravalent molecules are desired but Fc is not required, including antibodies (e.g., US Patent Publication Nos. 2005-0079170, 2007-0031436, 2010-0099853, European Patent Publication Nos. EP1078004, EP2371866, EP2361936 and EP1293514; PCT Publication Nos. WO 1999/057150, WO 2003/025018 , and WO2013/013700).
IV. ADAM9

A representative human ADAM9 polypeptide (NCBI sequence NP_003807, including a 28 amino acid residue signal sequence, underlined) has the amino acid sequence (SEQ ID NO:5):
MGSGARFPSG TLRVRWLLLL GLVGPVLG AA RPGFQQTSHL SSYEIITPWR LTRERREAPR PYSKQVSYVI
QAEGKEHIIH LERNKDLLPE DFVVYTYNKE GTLITDHPNI QNHCHYRGYV EGVHNSSIAL SDCFGLRGLL
HLENASYGIE PLQNSSHFEH IIYRMDDVYK EPLKCGVSNK DIEKETAKDE EEEPPSMTQL LRRRRAVLPQ
TRYVELFIVV DKERYDMMGR NQTAVREEMI LLANYLDSMY IMLNIRIVLV GLEIWTNGNL INIVGGAGDV
LGNFVQWREK FLITRRRHDS AQLVLKKGFG GTAGMAFVGT VCSRSHAGGI NVFGQITVET FASIVAHELG
HNLGMNHDDG RDCSCGAKSC IMNSGASGSR NFSSCSAEDF EKLTLNKGGN CLLNIPKPDE AYSAPSCGNK
LVDAGEECDC GTPKECELDP CCEGSTCKLK SFAECAYGDC CKDCRFLPGG TLCRGKTSEC DVPEYCNGSS
QFCQPDVFIQ NGYPCQNNKA YCYNGMCQYY DAQCQVIFGS KAKAAPKDCF IEVNSKGDRF GNCGFSGNEY
KKCATGNALC GKLQCENVQE IPVFGIVPAI IQTPSRGTKC WGVDFQLGSD VPDPGMVNEG TKCGAGKICR
NFQCVDASVL NYDCDVQKKC HGHGVCNSNK NCHCENGWAP PNCETKGYGG SVDSGPTYNE MNTALRDGLL
VFFFLIVPLI VCAIFIFIKR DQLWRSYFRK KRSQTYESDG KNQANPSRQP GSVPRHVSPV TPPREVPIYA
NRFAVPTYAA KQPQQFPSRP PPPQPKVSSQ GNLIPARPAP APPLYSSLT
have
Of the 819 amino acid residues of ADAM9 (SEQ ID NO: 5), residues 1-28 are the signal sequence, residues 29-697 are the extracellular domain, residues 698-718 are the transmembrane domain, and residues 719-819 are the intracellular domain. Three structural domains are located within the extracellular domain: the Reprolysin (M12B) family zinc metalloprotease domain (approximately residues 212-406); the disintegrin domain (approximately residues 423-497); and the EGF-like domain (approximately residues Groups 644-697). A number of post-translational modifications and isoforms have been identified in which the protein is proteolytically cleaved in the trans-Golgi network before it reaches the plasma membrane to generate the mature protein. Removal of the prodomain occurs by cleavage at two different sites. It is likely processed by proprotein convertases such as furin at the interface between the pro- and catalytic domains (Arg-205/Ala-206). An additional upstream cleavage proprotein convertase site (Arg-56/Glu-57) has an important role in the activation of ADAM9.

A representative cynomolgus monkey ADAM9 polypeptide (NCBI sequence XM_005563126.2, including a possible 28 amino acid residue signal sequence, underlined) has the amino acid sequence (SEQ ID NO:6):
MSGSVGSPSG TLRVRWLLLL CLVGPVLG AA RPGFQQTSHL SSYEIITPWR LTRERREAPR PYSKQVSYLI
QAEGKEHIIH LERNKDLLPE DFVVYTYNKE GTVITDHPNI QNHCHFRGYV EGVYNSSVAL SNCFGLRGLL
HLENASYGIE PLQNSSHFEH IIYRMDDVHK EPLKCGVSNK DIEKETTKDE EEEPPSMTQL LRRRRAVLPQ
TRYVELFIVV DKERYDMMGR NQTAVREEMI LLANYLDSMY IMLNIRIVLV GLEIWTNGNL INIAGGAGDV
LGNFVQWREK FLITRRRHDS AQLVLKKGFG GTAGMAFVGT VCSRSHAGGI NVFGHITVET FASIVAHELG
HNLGMNHDDG RDCSCGAKSC IMNSGASGSR NFSSCSAEDF EKLTLNKGGN CLLNIPKPDE AYSAPSCGNK
LVDAGEECDC GTPKECELDP CCEGSTCKLK SFAECAYGDC CKDCRFLPGG TLCRGKTSEC DVPEYCNGSS
QFCQPDVFIQ NGYPCQNNKA YCYNGMCQYY DAQCQVIFGS KAKAAPKDCF IEVNSKGDRF GNCGFSGNEY
KKCATGNALC GKLQCENVQE IPVFGIVPAI IQTPSRGTKC WGVDFQLGSD VPDPGMVNEG TKCGADKICR
NFQCVDASVL NYDCDIQKKC HGHGVCNSNK NCHCENGWAP PNCETKGYGG SVDSGPTYNE MNTALRDGLL
VFFFLIVPLI VCAIFIFIKR DQLWRRYFRK KRSQTYESDG KNQANPSRQP VSVPRHVSPV TPPREVPIYA
NRFPVPTYAA KQPQQFPSRP PPPQPKVSSQ GNLIPARPAP APPLYSSLT
have
The Reprolysin (M12B) family zinc metalloprotease domain of the protein is about residues 212-406; the disintegrin domain of the protein is about residues 423-497.

In certain embodiments, the ADAM9 binding molecules (e.g., scFvs, antibodies, bispecific diabodies, etc.) of the invention are any one, two, three, four of the following categories: Characterized by 5, 6, 7, or 8:
(1) the ability to immunospecifically bind to human ADAM9 endogenously expressed on the surface of cancer cells;
(2) specifically binds human and non-human primate ADAM9 (e.g., cynomolgus monkey ADAM9) with similar binding affinities;
(3) specifically binds human ADAM9 with an equilibrium binding constant (K _D ) of 4 nM or less;
(4) specifically binds non-human primate ADAM9 with an equilibrium binding constant (K _D ) of 4 nM or less;
(5) specifically binds to human ADAM9 at an on-rate (ka) of 5×10 ⁵ M ⁻¹ min ⁻¹ or more;
(6) specifically binds to non-human primate ADAM9 at an on rate (ka) of 1×10 ⁶ M ⁻¹ min ⁻¹ or more;
(7) specifically binds to human ADAM9 with an off-rate (kd) of 1×10 ⁻³ min ⁻¹ or less;
(8) specifically binds to non-human primate ADAM9 with an off-rate (kd) of 9×10 ⁻⁴ min ⁻¹ or less;
(9) at least a 100-fold enhancement (e.g., at least 100-fold, at least 150-fold) in binding affinity for cynomolgus monkey ADAM9 (e.g., as measured by BIACORE® analysis) compared to the chimeric or murine parental antibody; , at least 200-fold, at least 250-fold, at least 300-fold, at least 350-fold, at least 400-fold, at least 450-fold, at least 500-fold, at least 550-fold, or at least 600-fold enhancement) and optimized to retain high affinity binding to human ADAM9 (eg, as measured by BIACORE® analysis).

As described herein, binding constants for ADAM9 binding molecules can be determined using surface plasmon resonance, eg, by BIACORE® analysis. The surface plasmon resonance data can be fitted to a 1:1 Langmuir binding model (simultaneous kakd) and the equilibrium binding constant K _D can be calculated from the ratio of the rate constants kd/ka. Such binding constants may be monovalent ADAM9 binding molecules (i.e. molecules containing a single ADAM9 epitope binding site), bivalent ADAM9 binding molecules (i.e. molecules containing two ADAM9 epitope binding sites), or higher valences. (eg, molecules containing three, four, or more ADAM9 epitope binding sites).

The invention specifically provides ADAM9 binding molecules comprising anti-ADAM9 light chain variable (VL) domain(s) and anti-ADAM9 heavy chain variable (VH) domain(s) that immunospecifically bind to an epitope of a human ADAM9 polypeptide. (eg, antibodies, diabodies, trivalent binding molecules, etc.) are specifically included. Unless otherwise stated, all such ADAM9 binding molecules are capable of immunospecifically binding human ADAM9. As used herein, such ADAM9 variable domains are referred to as "anti-ADAM9-VL" and "anti-ADAM9-VH," respectively.
V. Murine anti-human ADAM9 antibody

A murine anti-ADAM9 antibody that blocks ADAM9's target protein processing activity, is internalized, and has anti-tumor activity has been identified (see, eg, US Pat. No. 8,361,475). This antibody, designated as the "anti-KID24" antibody produced by hybridoma clone ATCC PTA-5174 in US Pat. Nos. 7,674,619 and 8,361,475, is herein referred to as "MAB -A”. MAB-A shows strong preferential binding to tumor over normal tissue (see Figures 7A-7C). MAB-A showed little or no staining across a large panel of normal cell types (Table 1).

As shown in Figures 8A-8B, MAB-A binds human ADAM9 with high affinity, but binds non-human primate (eg, cynomolgus monkey) ADAM9 to a lesser extent.

The amino acid sequences of the VL and VH domains of MAB-A are provided below. The VH and VL domains of MAB-A were humanized and the CDRs optimized to improve affinity and/or remove potential amino acid trends. CDR _H3 was further optimized to enhance binding to non-human primate ADAM9 while maintaining its high affinity to human ADAM9.

A preferred anti-human ADAM9 binding molecule of the invention is one, two or all three of the CDR _Hs of the VH domain and/or one, two or all of the CDR _Ls of the VL domain of an optimized variant of MAB-A. It has all three and preferably also has the humanized framework regions (“FR”) of the VH and/or VL domains of humanized MAB-A. Other preferred anti-human ADAM9 binding molecules of the invention have the entire VH and/or VL domains of a humanized/optimized variant of MAB-A. Such preferred anti-human ADAM9 binding molecules include antibodies, bispecific (or multispecific) antibodies, chimeric or humanized antibodies, BiTe, diabodies, etc., as well as naturally occurring G or A variant Fc regions. Included are such binding molecules further comprising

The invention inter alia:
(A) (1) the three CDR _Hs of the VH domain of MAB-A; and (2) the four FRs of the VH domain of the humanized variant of MAB-A; or (B) (1) the VL of MAB-A. and (2) the four FRs of the VL domain of the humanized variant of MAB-A; or ( _C ) the three CDR _H of the VH domain of the optimized variant of MAB-A; and MAB - 3 CDR _L of the VL domain of A; or (D) 3 CDR _H of the VH domain of MAB-A; and 3 CDR _L of the optimized variant MAB-A VL domain; or (E) MAB- 3 CDR _H of the VH domain of the optimized variant of A; and 3 CDR _L of the VL domain of the optimized MAB-A; or (F) (1) 3 of the VH domain of the optimized variant of MAB-A and (2) the four FRs of the _VH domain of the humanized variant of MAB-A; or (G) (1) the three CDRs of the _VL domain of the optimized variant of MAB-A; and ( 2) the four FRs of the VL domain of a humanized variant of MAB-A; or (H)(1) the VH domain of a humanized/optimized variant of MAB-A; and (2) humanized MAB-A. /ADAM9-binding molecules comprising an ADAM9-binding domain with an optimized variant of the VL domain. Murine antibody "MAB-A"

The amino acid sequence of the VH domain of the murine anti-ADAM9 antibody MAB-A is SEQ ID NO:7 (CDR _H residues are underlined):
QVQLQQPGAE LVKPGASVKL SCKASGYTFT SYWMH WVKQR PGQGLEWIG E IIPINGHTNY NEKFKS KATL
TLDKSSSTAY MQLSSLASED SAVYYCAR GG YYYYGSRDYF DY WGQGTTLT VSS
is.

The amino acid sequence of the CDR _H 1 domain of MAB-A is (SEQ ID NO:8): SYWMH.

The amino acid sequence of the CDR _H2 domain of MAB-A is (SEQ ID NO:9): EIIPINGHTNYNEKFKS.

The amino acid sequence of the CDR _H 3 domain of MAB-A is (SEQ ID NO: 10): GGYYYYGSRDYFDY.

The amino acid sequence of the VL domain of the murine anti-ADAM9 antibody MAB-A is SEQ ID NO: 11 (CDR _L residues are underlined):
DIVLTQSPAS LAVSLGQRAT ISC KASQSVD YDGDSYMN WY QQIPGQPPKL LIY AASDLES GIPARFSGSG
SGTDFTLNIH PVEEEDAATY YC QQSHEDPF T FGGGTKLEI K
is.

The amino acid sequence of the CDR _L 1 domain of MAB-A is (SEQ ID NO: 12): KASQSVDYDGDSYMN.

The amino acid sequence of the CDR _L2 domain of MAB-A is (SEQ ID NO: 13): AASDLES.

The amino acid sequence of the CDR _L 3 domain of MAB-A is (SEQ ID NO: 14): QQSHEDPFT.
VI. Exemplary Humanized/Optimized Anti-ADAM9-VH and VL Domains1. Mutant VH domains of MAB-A

Certain preferred humanized/optimized anti-ADAM9-VH domain amino acid sequences of MAB-A are variants of the VH domain of ADAM9-MAB-A (SEQ ID NO: 7) and SEQ ID NO: 15 (CDR _H residues are underlined), represented by:
EVQLVESGGG LVKPGGSLRL _SCAASGFTFS SYWX 1H WVRQA
PGKGLEWVG E IIPIX ₂ GHTNY NEX ₃ FX ₄ X ₅ RFTI SLDNSKNTLY
LQMGSLRAED _TAVYYCAR GG YYYYX _{6X7X8X9X10X11} DYWGQGTTVT _{_} _ _{_} _ _{_} _ _{_} _
VSS
wherein: X ₁ , X ₂ , X ₃ , X ₄ , X ₅ and X ₆ are independently selected;
X ₁ is M or I; X ₂ is N or F;
X ₃ is K or R; X ₄ is K or Q;
X ₅ is S or G and X ₆ is P, F, Y, W, I, L, V, T, G or D;
X ₇ , X ₈ , X ₉ , X ₁₀ and X ₁₁ are:
(A) When _X6 is P: _X7 is K or R; _X8 is F or M; _X9 is G; _X10 is W or F; and _X11 is M, L or K;
(B) when X ₆ is F, Y or W: X ₇ is N or H; X ₈ is S or K; _{X 9} is G or A; X ₁₀ is T or V; or K;
(C) when X ₆ is I, L or V: X ₇ is G; X ₈ is K; X ₉ is G or A; X ₁₀ is V; and X ₁₁ is M, L or K;
(D) when X6 is T: _X7 is G; _X8 is K, M or N; _X9 is G; _X10 is _V or T; and _X11 is L or M;
(E) where X ₆ is G: X ₇ is G; X ₈ is S; X ₉ is G; X ₁₀ is V; and X ₁₁ is L;
(F) When X6 is D: _X7 is S; _X8 is N; _X9 is A; _X10 is _V ; and _X11 is L
is selected so that

Preferred Humanized Anti-ADAM9 VH Domain of MAB-A: Amino Acid Sequences of hMAB-A VH(1) (SEQ ID NO: 16) and Certain Preferred Humanized/Optimized Anti-ADAM9-VH Domains of MAB-A:
hMAB-A VH(2) (SEQ ID NO: 17)
hMAB-A VH(3) (SEQ ID NO: 18)
hMAB-A VH(4) (SEQ ID NO: 19)
hMAB-A VH(2A) (SEQ ID NO:20)
hMAB-A VH(2B) (SEQ ID NO:21)
hMAB-A VH(2C) (SEQ ID NO:22)
hMAB-A VH(2D) (SEQ ID NO:23)
hMAB-A VH(2E) (SEQ ID NO:24)
hMAB-A VH(2F) (SEQ ID NO: 25)
hMAB-A VH(2G) (SEQ ID NO: 26)
hMAB-A VH(2H) (SEQ ID NO:27)
hMAB-A VH(2I) (SEQ ID NO:28) and hMAB-A VH(2J) (SEQ ID NO:29)
are presented below (CDR _H residues are single underlined; differences to hMAB-A VH(1) (SEQ ID NO: 7) are double underlined).
hMAB-A VH(1) (SEQ ID NO: 16):
EVQLVESGGG LVKPGGSLRL SCAASGFTFS SYWMH WVRQA PGKGLEWVG E IIPINGHTNY NEKF KSRFTI
SLDNSKNTLY LQMGSLRAED TAVYYCAR GG YYYYGSRDYF DYWGQGTTVT VSS

hMAB-A VH(2) (SEQ ID NO: 17):
EVQLVESGGG LVKPGGSLRL SCAASGFTFS SYWMH WVRQA PGKGLEWVG E IIPIFGHTNY NEKFKS RFTI
SLDNSKNTLY LQMGSLRAED TAVYYCAR GG YYYYGSRDYF DYWGQGTTVT VSS

hMAB-A VH(3) (SEQ ID NO: 18):
EVQLVESGGG LVKPGGSLRL SCAASGFTFS SYWMH WVRQA PGKGLEWVG E IIPIFGHTNY NERFQG RFTI
SLDNSKNTLY LQMGSLRAED TAVYYCAR GG YYYYGSRDYF DYWGQGTTVT VSS

hMAB-A VH(4) (SEQ ID NO: 19):
EVQLVESGGG LVKPGGSLRL SCAASGFTFS SYWIH WVRQA PGKGLEWVG E IIPIFGHTNY NERFQG RFTI
SLDNSKNTLY LQMGSLRAED TAVYYCAR GG YYYYGSRDYF DYWGQGTTVT VSS

hMAB-A VH(2A) (SEQ ID NO: 20):
EVQLVESGGG LVKPGGSLRL SCAASGFTFS SYWMH WVRQA PGKGLEWVG E IIPIFGHTNY NEKFKS RFTI
SLDNSKNTLY LQMGSLRAED TAVYYCAR GG YYYYFNSGTL DYWGQGTTVT VSS

hMAB-A VH(2B) (SEQ ID NO:21):
EVQLVESGGG LVKPGGSLRL SCAASGFTFS SYWMH WVRQA PGKGLEWVG E IIPIFGHTNY NEKFKS RFTI
SLDNSKNTLY LQMGSLRAED TAVYYCAR GG YYYYIGKGVL DYWGQGTTVT VSS

hMAB-A VH(2C) (SEQ ID NO:22):
EVQLVESGGG LVKPGGSLRL SCAASGFTFS SYWMH WVRQA PGKGLEWVG E IIPIFGHTNY NEKFKS RFTI
SLDNSKNTLY LQMGSLRAED TAVYYCAR GG YYYYPRFGWL DY WGQGTTVT VSS

hMAB-A VH(2D) (SEQ ID NO:23):
EVQLVESGGG LVKPGGSLRL SCAASGFTFS SYWMH WVRQA PGKGLEWVG E IIPIFGHTNY NEKFKS RFTI
SLDNSKNTLY LQMGSLRAED TAVYYCAR GG YYYYTGKGVL DYWGQGTTVT VSS

hMAB-A VH(2E) (SEQ ID NO:24):
EVQLVESGGG LVKPGGSLRL SCAASGFTFS SYWMH WVRQA PGKGLEWVG E IIPIFGHTNY NEKFKS RFTI
SLDNSKNTLY LQMGSLRAED TAVYYCAR GG YYYYDSNAVL DYWGQGTTVT VSS

hMAB-A VH(2F) (SEQ ID NO: 25):
EVQLVESGGG LVKPGGSLRL SCAASGFTFS SYWMH WVRQA PGKGLEWVG E IIPIFGHTNY NEKFKS RFTI
SLDNSKNTLY LQMGSLRAED TAVYYCAR GG YYYYFHSGTL DY WGQGTTVT VSS

hMAB-A VH(2G) (SEQ ID NO:26):
EVQLVESGGG LVKPGGSLRL SCAASGFTFS SYWMH WVRQA PGKGLEWVG E IIPIFGHTNY NEKFKS RFTI
SLDNSKNTLY LQMGSLRAED TAVYYCAR GG YYYYFNKAVL DY WGQGTTVT VSS

hMAB-A VH(2H) (SEQ ID NO:27):
EVQLVESGGG LVKPGGSLRL SCAASGFTFS SYWMH WVRQA PGKGLEWVG E IIPIFGHTNY NEKFKS RFTI
SLDNSKNTLY LQMGSLRAED TAVYYCAR GG YYYYGGSGVL DYWGQGTTVT VSS

hMAB-A VH(2I) (SEQ ID NO:28):
EVQLVESGGG LVKPGGSLRL SCAASGFTFS SYWMH WVRQA PGKGLEWVG E IIPIFGHTNY NEKFKS RFTI
SLDNSKNTLY LQMGSLRAED TAVYYCAR GG YYYYPRQGFL DYWGQGTTVT VSS

hMAB-A VH(2J) (SEQ ID NO:29):
EVQLVESGGG LVKPGGSLRL SCAASGFTFS SYWMH WVRQA PGKGLEWVG E IIPIFGHTNY NEKFKS RFTI
SLDNSKNTLY LQMGSLRAED TAVYYCAR GG YYYYYNSGTL DY WGQGTTVT VSS

A preferred human amino acid sequence of the FRs of the anti-ADAM9-VH domain of humanized and/or optimized MAB-A is:
FR _H 1 domain (SEQ ID NO: 30): EVQLVESGGGLVKPGGSLRLSCAASGFTFS
FR _H2 domain (SEQ ID NO:31): WVRQAPGKGLEWVG
FR _H3 domain (SEQ ID NO:32): RFTISLDNSKNTLYLQMGSLRAEDTAVYYCAR
FR H4 domain (SEQ ID NO:33): _WGQGTTVTVSS
is.

Another preferred amino acid sequence of the CDR _H 1 domain of the anti-ADAM9-VH domain of MAB-A includes:
SEQ ID NO: 8: SYWMH
SEQ ID NO:34: SYWIH
is included.

Another preferred amino acid sequence of the CDR _H 2 domain of the anti-ADAM9-VH domain of MAB-A includes:
SEQ ID NO: 9: EIIPINGHTNYNEKFKS
SEQ ID NO: 35: EIIPIFGHTNYNEKFKS
SEQ ID NO: 36: EIIPIFGHTNYNERFQG
is included.

Another preferred amino acid sequence of the CDR _H 3 domain of the anti-ADAM9-VH domain of MAB-A includes:
SEQ ID NO: 10: GGYYYYGSRDYFDY
SEQ ID NO: 37: GGYYYYFNSGTLDY
SEQ ID NO: 38: GGYYYYIGKGVLDY
SEQ ID NO: 39: GGYYYYPRFGWLDY
SEQ ID NO: 40: GGYYYYTGKGVLDY
SEQ ID NO: 41: GGYYYYDSNAVLDY
SEQ ID NO: 42: GGYYYYFHSGTLDY
SEQ ID NO: 43: GGYYYYFNKAVLDY
SEQ ID NO: 44: GGYYYYGGSGVLDY
SEQ ID NO: 45: GGYYYYPRQGFLDY
SEQ ID NO: 46: GGYYYYYNSGTLDY
is included.

Accordingly, the present invention:
(1) amino acid sequence:
SEQ ID NO: 47: SYWX ₁ H: X ₁ is M or I;
(2) _amino acid sequence:
SEQ ID NO: ₄₈ : _{EIIPIX2GHTNYNEX3FX4X5} : X2 _{, X3 , X4 ,} and _{X5 are} independently selected: X2 is _N or F; X3 is K or R; X ₄ is K or Q; X ₅ is S or _G and (3) amino acid sequence:
SEQ ID NO: 49: GGYYYYX ₆ X ₇ X ₈ X ₉ X ₁₀ X ₁₁ DY: X ₆ is P, F, Y, W, I, L, V, T, G or D, and X ₇ , X ₈ , X ₉ , X ₁₀ and X ₁₁ are:
(A) When _X6 is P: _X7 is K or R; _X8 is F or M; _X9 is G; _X10 is W or F; and _X11 is M, L or K;
(B) when X ₆ is F, Y or W: X ₇ is N or H; X ₈ is S or K; _{X 9} is G or A; X ₁₀ is T or V; or K;
(C) when X ₆ is I, L or V: X ₇ is G; X ₈ is K; X ₉ is G or A; X ₁₀ is V; and X ₁₁ is M, L or K;
(D) when X6 is T: _X7 is G; _X8 is K, M or N; _X9 is G; _X10 is _V or T; and _X11 is L or M;
(E) if X ₆ is G: X ₇ is G; X ₈ is S; X ₉ is G; _{X 10} is _V ; X ₈ is _N ; X ₉ is A; _{X 10 is V} ;

A first exemplary humanized/optimized IgG1 heavy chain of a derivative/variant of MAB-A comprises the hMAB-A VH(2) domain (SEQ ID NO: 17) and has an amino acid sequence (SEQ ID NO: 50):
EVQLVESGGG LVKPGGSLRL SCAASGFTFS SYWMHWVRQA PGKGLEWVGE IIPIFGHTNY NEKFKSRFTI
SLDNSKNTLY LQMGSLRAED TAVYYCARGG YYYYGSRDYF DYWGQGTTVT VSSASTKGPS VFPLAPSSKS
TSGGTAALGC LVKDYFPEPV TVSWNSGALT SGVHTFPAVL QSSGLYSLSS VVTVPSSSLG TQTYICNVNH
KPSNTKVDKR VEPKSCDKTH TCPPCPAPEL LGGPSVFLFP PKPKDTLMIS RTPEVTCVVV DVSHEDPEVK
FNWYVDGVEV HNAKTKPREE QYNSTYRVVS VLTVLHQDWL NGKEYKCKVS NKALPAPIEK TISKAKGQPR
EPQVYTLPPS REEMTKNQVS LTCLVKGFYP SDIAVEWESN GQPENNYKTT PPVLDSDGSF FLYSKLTVDK
SRWQQGNVFS CSVMHEALHN HYTQKSLSLS
PG X
where X is lysine (K) or absent.

A second exemplary humanized/optimized IgG1 heavy chain of a derivative/variant of MAB-A comprises the hMAB-A VH(2C) domain (SEQ ID NO:22) and has the amino acid sequence (SEQ ID NO:51):
EVQLVESGGG LVKPGGSLRL SCAASGFTFS SYWMHWVRQA PGKGLEWVGE IIPIFGHTNY NEKFKSRFTI
SLDNSKNTLY LQMGSLRAED TAVYYCARGG YYYYPRFGWL DYWGQGTTVT VSSASTKGPS VFPLAPSSKS
TSGGTAALGC LVKDYFPEPV TVSWNSGALT SGVHTFPAVL QSSGLYSLSS VVTVPSSSLG TQTYICNVNH
KPSNTKVDKR VEPKSCDKTH TCPPCPAPEL LGGPSVFLFP PKPKDTLMIS RTPEVTCVVV DVSHEDPEVK
FNWYVDGVEV HNAKTKPREE QYNSTYRVVS VLTVLHQDWL NGKEYKCKVS NKALPAPIEK TISKAKGQPR
EPQVYTLPPS REEMTKNQVS LTCLVKGFYP SDIAVEWESN GQPENNYKTT PPVLDSDGSF FLYSKLTVDK
SRWQQGNVFS CSVMHEALHN HYTQKSLSLS
PG X
where X is lysine (K) or absent.

A third exemplary humanized/optimized IgG1 heavy chain of a derivative/variant of MAB-A comprises the hMAB-A VH(2I) domain (SEQ ID NO:28) and has the amino acid sequence (SEQ ID NO:52):
EVQLVESGGG LVKPGGSLRL SCAASGFTFS SYWMHWVRQA PGKGLEWVGE IIPIFGHTNY NEKFKSRFTI
SLDNSKNTLY LQMGSLRAED TAVYYCARGG YYYYPRQGFL DYWGQGTTVT VSSASTKGPS VFPLAPSSKS
TSGGTAALGC LVKDYFPEPV TVSWNSGALT SGVHTFPAVL QSSGLYSLSS VVTVPSSSLG TQTYICNVNH
KPSNTKVDKR VEPKSCDKTH TCPPCPAPEL LGGPSVFLFP PKPKDTLMIS RTPEVTCVVV DVSHEDPEVK
FNWYVDGVEV HNAKTKPREE QYNSTYRVVS VLTVLHQDWL NGKEYKCKVS NKALPAPIEK TISKAKGQPR
EPQVYTLPPS REEMTKNQVS LTCLVKGFYP SDIAVEWESN GQPENNYKTT PPVLDSDGSF FLYSKLTVDK
SRWQQGNVFS CSVMHEALHN HYTQKSLSLS
PG X
where X is lysine (K) or absent.

As provided herein, the CH2-CH3 domains of the Fc region can be engineered to, for example, reduce effector function. In certain embodiments, the CH2-CH3 domains of exemplary humanized/optimized IgG1 heavy chains of the invention comprise one or more substitutions selected from: L234A and L235A.

Thus, a fourth exemplary humanized/optimized IgG1 heavy chain of a derivative/variant of MAB-A comprises the hMAB-A VH(2I) domain (SEQ ID NO: 28) and further CH2-CH3 containing substitutions L234A and L235A (SEQ ID NO: 106) in the domain (underlined below), the amino acid sequence (SEQ ID NO: 202):
EVQLVESGGG LVKPGGSLRL SCAASGFTFS SYWMHWVRQA PGKGLEWVGE IIPIFGHTNY NEKFKSRFTI
SLDNSKNTLY LQMGSLRAED TAVYYCARGG YYYYPRQGFL DYWGQGTTVT VSSASTKGPS VFPLAPSSKS
TSGGTAALGC LVKDYFPEPV TVSWNSGALT SGVHTFPAVL QSSGLYSLSS VVTVPSSSLG TQTYICNVNH
KPSNTKVDKR VEPKSCDKTH TCPPCPAPE A A GGPSVFLFP PKPKDTLMIS RTPEVTCVVV DVSHEDPEVK
FNWYVDGVEV HNAKTKPREE QYNSTYRVVS VLTVLHQDWL NGKEYKCKVS NKALPAPIEK TISKAKGQPR
EPQVYTLPPS REEMTKNQVS LTCLVKGFYP SDIAVEWESN GQPENNYKTT PPVLDSDGSF FLYSKLTVDK
SRWQQGNVFS CSVMHEALHN HYTQKSLSLS
PG X
where X is lysine (K) or absent.
2. Mutant VL domains of MAB-A

A preferred humanized/optimized anti-ADAM9-VL domain amino acid sequence of MAB-A is a variant of the VL domain of ADAM9-MAB-A (SEQ ID NO: 11) and SEQ ID NO: 53 (CDR _L residues are underlined). ):
DIVMTQSPDS LAVSLGERAT ISC X ₁₂ ASQSVD YX ₁₃ GDSYX ₁₄ N WY
QQKPGQPPKL LIY AASDLES GIPARFSGSG SGTDFTLTIS
SLEPEDFATY YC QQSX _15X16X17PF T _FGQGTKLEIK _ _{_}
wherein: X ₁₂ , X ₁₃ , X ₁₄ , X ₁₅ , X ₁₆ , and X ₁₇ are independently selected and: _{X 12} is K or R; _Yes ; X ₁₄ is M or L; X ₁₅ is H or Y; X ₁₆ is E or S;

Preferred humanized anti-ADAM9-MAB-A VL domain amino acid sequences: hMAB-A VL(1) (SEQ ID NO:54), and MAB-A: hMAB-A VL(2) (SEQ ID NO:55), hMAB-A The amino acid sequences of certain preferred humanized/optimized anti-ADAM9-VL domains of VL(3) (SEQ ID NO:56) and hMAB-A VL(4) (SEQ ID NO:57) are provided below (CDR _L residues Groups are single underlined; differences to hMAB-A VL(1) (SEQ ID NO: 54) are double underlined).
hMAB-A VL(1) (SEQ ID NO:54):
DIVMTQSPDS LAVSLGERAT ISC KASQSVD YDGDSYMN WY QQKPGQPPKL LIY AASDLES GIPARFSGSG
SGTDFTLTIS SLEPEDFATY YC QQSHEDPF T FGQGTKLEI K
hMAB-A VL(2) (SEQ ID NO:55):
DIVMTQSPDS LAVSLGERAT ISC KASQSVD YSGDSYMN WY QQKPGQPPKL LIY AASDLES GIPARFSGSG
SGTDFTLTIS SLEPEDFATY YC QQSHEDPF T FGQGTKLEI K
hMAB-A VL(3) (SEQ ID NO:56):
DIVMTQSPDS LAVSLGERAT ISC RASQSVD YSGDSYMN WY QQKPGQPPKL LIY AASDLES GIPARFSGSG
SGTDFTLTIS SLEPEDFATY YC QQSHEDPF T FGQGTKLEI K
hMAB-A VL(4) (SEQ ID NO:57):
DIVMTQSPDS LAVSLGERAT ISC RASQSVD YSGDSYLN WY QQKPGQPPKL LIY AASDLES GIPARFSGSG
SGTDFTLTIS SLEPEDFATY YC QQSYSTPF T FGQGTKLEI K

Accordingly, a preferred human amino acid sequence of the FRs of the VL domain of humanized and/or optimized anti-ADAM9-MAB-A is:
FR _L 1 domain (SEQ ID NO: 58): DIVMTQSPDSLAVSLGERATISC
FR _L2 domain (SEQ ID NO:59): WYQQKPGQPPKLLIY
FR _L 3 domain (SEQ ID NO: 60): GIPARFSGSGSGTDFTLTISSLEPEDFATYYC
FR _L 4 domain (SEQ ID NO: 61): FGQGTKLEIK
is.

Another preferred amino acid sequence of the CDR _L 1 domain of the anti-ADAM9-VL domain is:
SEQ ID NO: 12: KASQSVDYDGDSYMN
SEQ ID NO: 62: KASQSVDYSGDSYMN
SEQ ID NO: 63: RASQSVDYSGDSYMN
SEQ ID NO: 64: RASQSVDYSGDSYLN
including.

Another preferred amino acid sequence of the CDR _L 3 domain of the anti-ADAM9-VL domain is:
SEQ ID NO: 14: QQSHEDPFT
SEQ ID NO: 65: QQSYSTPFT
including.

Accordingly, the present invention:
( ₁ ) _Amino acid sequence: SEQ ID NO: 66: _{X12ASQSVDYX13GDSYX14N}
wherein: X ₁₂ , X ₁₃ , X ₁₄ are independently selected, and X ₁₂ is K or R; X ₁₃ is D or S; and X ₁₄ is M or _L ;
(2) amino acid sequence: SEQ ID NO: 13: AASDLES
and (3) amino acid sequence: SEQ ID NO: 67: _{QQSX 15} X ₁₆ X ₁₇ PFT
wherein: X ₁₅ , X ₁₆ and X ₁₇ are independently selected and X ₁₅ is H or Y; X ₁₆ is E or S; and X ₁₇ is D or _T. includes an anti-ADAM9 antibody VL domain containing

An exemplary humanized/optimized IgG1 light chain of a derivative/variant of MAB-A comprises the hMAB-A VL(2) domain (SEQ ID NO:55) and has the amino acid sequence (SEQ ID NO:68):
DIVMTQSPDS LAVSLGERAT ISCKASQSVD YSGDSYMNWY QQKPGQPPKL LIYAASDLES GIPARFSGSG
SGTDFTLTIS SLEPEDFATY YCQQSHEDPF TFGQGTKLEI KRTVAAPSVF IFPPSDEQLK SGTASVVCLL
NNFYPREAKV QWKVDNALQS GNSQESVTEQ DSKDSTYSLS STLTLSKADY EKHKVYACEV THQGLSSPVT
KSFNRGEC
have

Accordingly, the invention further provides a MAB-A CDR _H 1, CDR _H 2, CDR _H 3, CDR _L 1, CDR _L 2, or CDR _L 3 immunospecifically binding to an epitope of the human ADAM9 polypeptide. and one of said MAB-A CDR _H 1, and said MAB-A CDR _H 2 and one of said MAB-A CDR _H 3 and one of said MAB-A CDR _L 1 and one of said MAB-A CDR _L 2 and said MAB _ADAM9 binding molecules comprising one of the -A CDR L3 are specifically envisaged.

The present invention provides an ADAM9-binding molecule further comprising any of said humanized MAB-A FR _H 1, FR _H 2, FR _H 3, or FR _H 4, FR _L 1, FR _L 2, FRL3, or FR _L 4. and specifically including FR _H 1, FR _H 2, FR _H 3, and FR _H 4, and/or including FR _L 1, FR _L 2, FR _L 3, FR _L 4, and FR _H 1 Assume a binding molecule.

In some embodiments, the ADAM9 binding molecule:
(a) SEQ ID NOs: 8, 35 and 10 and SEQ ID NOs: 62, 13 and 14, respectively;
(b) SEQ ID NOs: 8, 35 and 10 and SEQ ID NOs: 63, 13 and 14, respectively;
(c) SEQ ID NOs: 8, 36 and 10 and SEQ ID NOs: 63, 13 and 14, respectively;
(d) SEQ ID NOs: 34, 36 and 10 and SEQ ID NOs: 64, 13 and 65, respectively
(e) SEQ ID NOs: 8, 35 and 37 and SEQ ID NOs: 62, 13 and 14, respectively;
(f) SEQ ID NOs: 8, 35 and 38 and SEQ ID NOs: 62, 13 and 14, respectively;
(g) SEQ ID NOs: 8, 35 and 39 and SEQ ID NOs: 62, 13 and 14, respectively;
(h) SEQ ID NOs: 8, 35 and 40 and SEQ ID NOs: 62, 13 and 14, respectively;
(i) SEQ ID NOs: 8, 35 and 41 and SEQ ID NOs: 62, 13 and 14, respectively;
(j) SEQ ID NOs: 8, 35 and 42 and SEQ ID NOs: 62, 13 and 14, respectively;
(k) SEQ ID NOs: 8, 35 and 43 and SEQ ID NOs: 62, 13 and 14, respectively;
(l) SEQ ID NOs: 8, 35 and 44 and SEQ ID NOs: 62, 13 and 14, respectively;
(m) SEQ ID NOs:8, 35 and 45 and SEQ ID NOs:62, 13 and 14 respectively; and (n) SEQ ID NOs:8, 35 and 46 and SEQ ID NOs:62, 13 and 14 respectively
CDR _H 1 domain, CDR _H 2 domain, and CDR _H 3 domain and CDR _L 1 domain, CDR _L 2 domain, and CDR _L 3 domain, having a sequence selected from the group consisting of:

In certain embodiments, the ADAM9-binding molecule comprises CDR _H 1, CDR _H 2, and CDR _H 3 domains and CDRs having the sequences of SEQ ID NOs:8, 35 and 45 and SEQ ID NOs:62, 13 and 14, respectively. It contains an _L1 domain, a CDR _L2 domain, and a CDR _L3 domain.

In some embodiments, the ADAM9-binding molecule of the invention is a heavy chain variable domain (VH) having a sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence and a light chain variable domain (VL) containing:
SEQ ID NO: 17 and SEQ ID NO: 55, respectively;
SEQ ID NO: 17 and SEQ ID NO: 56, respectively;
SEQ ID NO: 18 and SEQ ID NO: 56, respectively;
SEQ ID NO: 19 and SEQ ID NO: 57, respectively;
SEQ ID NO: 20 and SEQ ID NO: 55, respectively;
SEQ ID NO: 21 and SEQ ID NO: 55, respectively;
SEQ ID NO: 22 and SEQ ID NO: 55, respectively;
SEQ ID NO: 23 and SEQ ID NO: 55, respectively;
SEQ ID NO: 24 and SEQ ID NO: 55, respectively;
SEQ ID NO: 25 and SEQ ID NO: 55, respectively;
SEQ ID NO: 26 and SEQ ID NO: 55, respectively;
SEQ ID NO: 27 and SEQ ID NO: 55, respectively;
SEQ ID NO:28 and SEQ ID NO:55, respectively; and SEQ ID NO:29 and SEQ ID NO:55, respectively.

By "substantially identical" or "identical" is meant a polypeptide that exhibits at least 50% identity to a reference amino acid sequence (e.g., any one of the amino acid sequences described herein). do. Preferably, such sequences are at least 60%, more preferably at least 80% or at least 85%, and more preferably at least 90%, at the amino acid level relative to the polypeptide sequences used for comparison. %, at least 95%, at least 99%, or even 100% identical.

配列同一性は、典型的には、配列分析ソフトウェア（例えば、Ｓｅｑｕｃｎｃｅ Ａｎａｌｙｓｉｓ Ｓｏｆｔｗａｒｅ Ｐａｃｋａｇｅ ｏｆ ｔｈｅ Ｇｅｎｅｔｉｃｓ Ｃｏｍｐｕｔｅｒ Ｇｒｏｕｐ，Ｕｎｉｖｅｒｓｉｔｙ ｏｆ Ｗｉｓｃｏｎｓｉｎ Ｂｉｏｔｅｃｈｎｏｌｏｇｙ Ｃｅｎｔｅｒ，１７１０ Ｕｎｉｖｅｒｓｉｔｙ Ａｖｅｎｕｅ，Ｍａｄｉｓｏｎ，Ｗｉｓ．５３７０５、ＢＬＡＳＴ、ＢＥＳＴＦＩＴ、ＧＡＰまたはＰＩＬＥＵＰ／ PRETTYBOX program). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and/or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary approach to determining the degree of identity, the BLAST program can be used, with probability scores from e-3 to e-100, denoting closely related sequences.

In certain embodiments, the ADAM9-binding molecules of the invention have a sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO:28 and SEQ ID NO:55, respectively. It includes a chain variable domain (VH) and a light chain variable domain (VL).

In certain embodiments, ADAM9 binding molecules of the invention comprise heavy and light chain sequences as follows:
SEQ ID NO: 50 and SEQ ID NO: 68, respectively;
SEQ ID NO: 51 and SEQ ID NO: 68, respectively;
SEQ ID NO:52 and SEQ ID NO:68, respectively; and SEQ ID NO:202 and SEQ ID NO:68, respectively.

The invention also provides ADAM9 binding molecules (e.g., antibodies, diabodies, trivalent binding molecules, etc.) are also explicitly envisioned. The present invention specifically contemplates ADAM9 binding molecules comprising any of the following combinations of humanized anti-ADAM9 VL or VH domains:

The invention specifically encompasses ADAM9 binding molecules comprising (i) the humanized/optimized anti-ADAM9-VL and/or VH domains described above and (ii) an Fc region. In certain embodiments, the ADAM9 binding molecule of the invention is a monoclonal antibody comprising (i) the humanized/optimized anti-ADAM9-VL and/or VH domains described above and (ii) an Fc region. In other embodiments, the ADAM9 binding molecules of the invention are: monoclonal antibodies, multispecific antibodies, synthetic antibodies, chimeric antibodies, single chain Fv (scFv), single chain antibodies, Fab fragments, F(ab') fragments, disulfides is selected from the group consisting of conjugated bispecific Fv (sdFv), BiTEs, diabodies, and trivalent binding molecules.

Although specific modifications to the anti-ADAM9 VH and VL domains are summarized above and compared in FIGS. 9A-9B, these modifications may be used when engineering the humanized and/or optimized anti-ADAM9-VH or VL domains of the invention. It is not necessary to modify most or all of the residues of The invention may also include minor alterations of these VH and VL sequences, including, for example, amino acid substitutions of C-terminal and/or N-terminal amino acid residues that can be introduced to facilitate subcloning.
VII. chimeric antigen receptor

ADAM9 binding molecules of the invention may be monospecific single chain molecules such as anti-ADAM9 single chain variable fragment (“anti-ADAM9-scFv”) or anti-ADAM9 chimeric antigen receptor (“anti-ADAM9-CAR”). . As described above, scFvs are made by linking the light and heavy chain variable domains together via a short connecting peptide. First generation chimeric antigen receptors (“CAR”) typically contain an intracellular domain from the CD3 zeta chain, which is the primary transmitter of signals from the endogenous T-cell receptor (“TCR”). Second-generation CARs contain additional intracellular signaling domains from various co-stimulatory protein receptors (e.g., CD28, 41BB, ICOS, etc.) fused to the cytoplasmic tail of the CAR to provide additional signals to T cells. have. Third generation CARs bind multiple signaling domains such as CD3ζ-CD28-41BB or CD3ζ-CD28-OX40 to further enhance their potency (Tettamanti, S. et al. (2013) “Targeting Of Acute Myeloid Leukemia By Cytokine-Induced Killer Cells Redirected With A Novel CD123-Specific Chimeric Antigen Receptor,” Br. J. Haematol. 161:389-401; Gill, S. et al. (2014) “Efficacy Against Human Acute Myeloid Leukemia And Myeloablation Of Normal Hematopoiesis In A Mouse Model Using Chimeric Antigen Receptor-Modified T Cells,” Blood 123(15): 2343-2354; Mardiros, A. et al. (2013) “T Cells Expressing CD123-Specific Chimeric Antigen Receptors Exhibit Specific Cytolytic Effector Functions And Antitumor Effects Against Human Acute Myeloid Leukemia,” Blood 122:3138-3148; Pizzitola, I. et al. ”Leukemia doi:10.1038/leu.2014.62). Chimeric antigen receptors are described in U.S. Patent Nos. 9,447,194; 9,266,960; 9,212,229; 9,074,000; and 8,465,743, and U.S. Patent Publication Nos. 2016/0272718, 2016/0144026, 2016/0130357, 2016/0081314, 2016/0075784, 2016/0058857, 2016/0046729, 2016/0046700, 2016/0045551, 2016/0015750, 2015/0320799, 2015/0307623, 2015/0307564, 2015/0038684, 2014/0134142 and 2013/0280285.

An anti-ADAM9-CAR of the invention comprises an anti-ADAM9-scFv fused to the intracellular domain of the receptor. The light chain variable (VL) domain and heavy chain variable (VH) domain of anti-ADAM9-scFv are selected from any of the humanized anti-ADAM9-VL and anti-ADAM9-VH domains disclosed herein. Preferably, the VH domains are: hMAB-A VH(1) (SEQ ID NO: 16), hMAB-A VH(2) (SEQ ID NO: 17), hMAB-A VH(3) (SEQ ID NO: 18), hMAB-A VH (4) (SEQ ID NO: 19), hMAB-A VH(2A) (SEQ ID NO: 20), hMAB-A VH(2B) (SEQ ID NO: 21), hMAB-A VH(2C) (SEQ ID NO: 22), hMAB- A VH(2D) (SEQ ID NO:23), hMAB-A VH(2E) (SEQ ID NO:24), hMAB-A VH(2F) (SEQ ID NO:25), hMAB-A VH(2G) (SEQ ID NO:26), selected from the group consisting of hMAB-A VH(2H) (SEQ ID NO:27), hMAB-A VH(2I) (SEQ ID NO:28), and hMAB-A VH(2J) (SEQ ID NO:29), wherein the VL domain is: hMAB-A VL(1) (SEQ ID NO: 54), hMAB-A VL(2) (SEQ ID NO: 55), hMAB-A VL(3) (SEQ ID NO: 56), and hMAB-A VL(4) (SEQ ID NO: 57). Combinations of humanized/optimized anti-ADAM9-VL and anti-ADAM9-VH domains and CDR _H s and CDR _L s combinations that can be used to form such chimeric antigen receptors are described above.

The intracellular domains of the anti-ADAM9-CAR of the invention are preferably: 41BB-CD3ζ, b2c-CD3ζ, CD28, CD28-4-1BB-CD3ζ, CD28-CD3ζ, CD28-FcεRIγ, CD28mut-CD3ζ, CD28-OX40- selected from the intracellular domain of any of CD3ζ, CD28-OX40-CD3ζ, CD3ζ, CD4-CD3ζ, CD4-FcεRIγ, CD8-CD3ζ, FceRIγ, FcεRIγCAIX, Heregulin-CD3ζ, IL-13-CD3ζ, or Ly49H-CD3ζ (Tettamanti, S. et al. (2013) “Targeting Of Acute Myeloid Leukaemia By Cytokine-Induced Killer Cells Redirected With A Novel CD123-Specific Chimeric Antigen Receptor,” Br. J. Haematol. 161:389-401; Gill, S. et al. (2014) "Efficacy Against Human Acute Myeloid Leukemia And Myeloablation Of Normal Hematopoiesis In A Mouse Model Using Chimeric Antigen Receptor-Modified T Cells," Blood 123(15): 2343-2354; Mardiros, A. et al. (2013) “T Cells Expressing CD123-Specific Chimeric Antigen Receptors Exhibit Specific Cytolytic Effector Functions And Antitumor Effects Against Human Acute Myeloid Leukemia,” Blood 122:3138-3148; Pizzitola, I. et al. (2014) “Chimeric Antigen Receptors Against CD33/CD123 Antigens Efficiently Target Primary Acute Myeloid Leukemia Cel ls in vivo,” Leukemia doi:10.1038/leu.2014.62).
VIII. Multispecific ADAM9 binding molecules

The present invention also provides an epitope binding site (preferably one, two or all three CDR Hs of an anti-ADAM9- _VH domain of the invention and/or one CDR _L of an anti-ADAM9-VL domain of the invention, two or all three or such anti-ADAM9-VH domains and/or such anti-ADAM9-VL domains) and a second epitope binding site that immunospecifically binds to a second epitope. Also of interest are multispecific (e.g., bispecific, trispecific, etc.) ADAM9 binding molecules that further comprise, wherein such second epitope is (i) a different epitope of ADAM9, or (ii) an epitope of a molecule that is not ADAM9. be. Such multispecific ADAM9 binding molecules preferably contain a combination of epitope binding sites that recognize a unique set of antigens for the target cell or tissue type. In particular, the present invention provides multispecific ADAM9 capable of binding epitopes of ADAM9 and molecules present on the surface of effector cells, particularly T lymphocytes, natural killer (NK) cells or other mononuclear cells. Regarding binding molecules. For example, such ADAM9 binding molecules of the invention can be constructed to contain an epitope binding site that immunospecifically binds to CD2, CD3, CD8, CD16, the T cell receptor (TCR), or NKG2D. .

One embodiment of the invention relates to a bispecific ADAM9 binding molecule capable of binding a "first epitope" and a "second epitope", wherein such epitopes are not identical to each other. Such bispecific molecules comprise a "VL1"/"VH1" domain capable of binding a first epitope and a "VL2"/"VH2" domain capable of binding a second epitope. The notations "VL1" and "VH1" refer to the light and heavy chain variable domains, respectively, that bind the "first" epitope of such bispecific molecules. Similarly, the notations "VL2" and "VH2" refer to the light and heavy chain variable domains, respectively, that bind the "second" epitope of such bispecific molecules. It is irrelevant whether a particular epitope is designated as first versus second epitope; such designation relates only to the presence and orientation of the domains of the polypeptide chain of the binding molecule of the invention. In one embodiment, one of such epitopes is an epitope of human ADAM9 and the other is a different epitope of ADAM9 or an epitope of a molecule that is not ADAM9. In certain embodiments, one such epitope is that of human ADAM9 and the other is a molecule present on the surface of effector cells such as T lymphocytes, natural killer (NK) cells or other mononuclear cells. For example, epitopes of CD2, CD3, CD8, CD16, T cell receptor (TCR), NKG2D, etc.). In certain embodiments, bispecific molecules comprise more than two epitope binding sites. Such bispecific molecules bind at least one epitope of ADAM9 and at least one epitope of a molecule that is not ADAM9, and may further bind an additional epitope of ADAM9 and/or an additional epitope of a molecule that is not ADAM9. can.

The invention particularly has epitope-binding fragments (e.g., VL and VH domains) of antibodies that enable them to coordinately bind to at least one epitope of ADAM9 and at least one epitope of a second molecule that is not ADAM9. Bispecific, trispecific and multispecific ADAM9 binding molecules (eg, bispecific antibodies, bispecific diabodies, trivalent binding molecules, etc.). The selection of the VL and VH domains of the polypeptide domains of such molecules is such that the polypeptide chains that make up such multispecific ADAM9 binding molecules are assembled to provide at least one specific for at least one epitope of ADAM9. It is arranged to form one functional epitope binding site and at least one functional epitope binding site specific for at least one epitope of a molecule that is not ADAM9. Preferably, the multispecific ADAM9 binding molecule comprises one, two or all three CDR Hs of an anti-ADAM9- _VH domain of the invention and/or an anti-ADAM9 binding molecule of the invention, as provided herein. - includes one, two or all three of the CDR _Ls of the VL domain, or such anti-ADAM9-VH domain and/or such anti-ADAM9-VL domain.
A. bispecific antibody

The present invention encompasses bispecific antibodies that are capable of simultaneously binding an epitope of ADAM9 and an epitope of a molecule that is not ADAM9. In some embodiments, the bispecific antibody capable of simultaneously binding ADAM9 and a second molecule that is not ADAM9 is PCT Publication No. WO 1998, each of which is hereby incorporated by reference in its entirety. /002463, WO2005/070966, WO2006/107786, WO2007/024715, WO2007/075270, WO2006/107617, WO2007/046893 WO 2007/146968, WO 2008/003103, WO 2008/003116, WO 2008/027236, WO 2008/024188, WO 2009/132876, International Publication No. 2009/018386, International Publication No. 2010/028797, International Publication No. 2010/028796, International Publication No. 2010/028795, International Publication No. 2010/108127, International Publication No. 2010/136172, International Publication No. WO 2011/086091, WO 2011/133886, WO 2012/009544, WO 2013/003652, WO 2013/070565, WO 2012/162583, WO 2012 /156430, WO2013/174873, and WO2014/022540.
B. Bispecific diabodies lacking the Fc region

one embodiment of the invention relates to a bispecific diabody capable of binding a first epitope and a second epitope, wherein the first epitope is an epitope of human ADAM9 and the second epitope is not ADAM9, Molecules (e.g., CD2, CD3, CD8, CD16, T cell receptor (TCR), NKG2D, etc., preferably present on the surface of effector cells such as T lymphocytes, natural killer (NK) cells or other mononuclear cells) ) is an epitope of Such diabodies are first poly(s) whose sequences allow the polypeptide chains to covalently bond to each other to form a covalently linked diabody capable of simultaneously binding an epitope of ADAM9 and a second epitope. It includes a peptide chain and a second polypeptide chain, and most preferably consists of such a first polypeptide chain and a second polypeptide chain.

The first polypeptide chain of such embodiments of the bispecific diabodies, in the N-terminal to C-terminal direction: N-terminal, first or second epitope (i.e., VL _{anti-ADAM9-VL} or VL _{epitope 2} ), a first intervening spacer peptide (linker 1), a second epitope (such first polypeptide chain comprises VL _{anti-ADAM9-VL} a VH domain of a monoclonal antibody capable of binding either to ADAM9 (if such first polypeptide chain comprises VL epitope 2), or to ADAM9 (if such first polypeptide chain comprises VL _{epitope 2} ), a second intervening It contains a spacer peptide (linker 2), a heterodimer-promoting domain and a C-terminus (Fig. 1).

The second polypeptide chain of this embodiment of the bispecific diabody is in the N-terminal to C-terminal direction: the N-terminal, the VL domain of a monoclonal antibody capable of binding each of the first or second epitopes (i.e. , VL _{anti-ADAM9-VL} or VL _{epitope 2} , which is the VL domain not selected for inclusion in the first polypeptide chain of the diabody), intervening spacer peptide (linker 1), second epitope Monoclonal capable of binding either (if such second polypeptide chain comprises VL _{anti-ADAM9-VL} ) or ADAM9 (if such second polypeptide chain comprises VL _{epitope 2} ) It comprises the VH domain of the antibody, a second intervening spacer peptide (linker 2) which may optionally contain a cysteine residue, the heterodimer promoting domain, and the C-terminus (Figure 1).

The VL domain of the first polypeptide chain interacts with the VH domain of the second polypeptide chain to form a first antigen specific for the first antigen (i.e., either ADAM9 or a molecule containing a second epitope). Forms a functional epitope binding site. Similarly, the VL domain of the second polypeptide chain forms a second functional epitope binding site specific for a second antigen (i.e. either the molecule containing the second epitope or the molecule containing ADAM9). To do so, it interacts with the VH domain of the first polypeptide chain. Thus, the selection of the VL and VH domains of the first and second polypeptide chains is such that the two polypeptide chains of the diabodies together are capable of binding both the ADAM9 epitope and the second epitope. VH domains (ie, they together comprise VL _{anti-ADAM9-VL} /VH _{anti-ADAM9-VH} and VL _{epitope 2} /VH _{epitope 2} ).

Most preferably, the length of the intervening spacer peptide (i.e., "linker 1" separating such VL and VH domains) substantially or completely prevents the VL and VH domains of the polypeptide chain from joining together. selected to prevent (eg, composed of 0, 1, 2, 3, 4, 5, 6, 7, 8 or 9 intervening linker amino group residues). Thus, the VL and VH domains of the first polypeptide chain are substantially or completely incapable of binding to each other. Similarly, the VL and VH domains of the second polypeptide chain are substantially or completely incapable of binding to each other. A preferred intervening spacer peptide (linker 1) has the sequence (SEQ ID NO: 69): GGGSGGGG.

The length and composition of the second intervening spacer peptide (“linker 2”) are selected based on the selection of one or more polypeptide domains that promote such dimerization (i.e., “heterodimer-promoting domains”). be done. Typically, the second intervening spacer peptide (linker 2) contains 3-20 amino acid residues. In particular, if the heterodimer-promoting domain(s) employed does not contain a cysteine residue, a second intervening spacer peptide (linker 2) containing cysteine is utilized. A second intervening spacer peptide containing cysteines (linker 2) contains 1, 2, 3 or more cysteines. A preferred cysteine-containing spacer peptide (linker 2) has the sequence GGCGGG (SEQ ID NO:70). Alternatively, linker 2 does not contain cysteines (e.g., GGG, GGGS (SEQ ID NO: 71), LGGGSG (SEQ ID NO: 72), GGGSGGGSGGG (SEQ ID NO: 73), ASTKG (SEQ ID NO: 74), LEPKSS (SEQ ID NO: 75), APSSS (SEQ ID NO: 76)), using a cysteine-containing heterodimer-promoting domain as described below. Optionally, both a cysteine-containing linker 2 and a cysteine-containing heterodimer promoting domain are used.

The heterodimer-promoting domain consists of GVEPKSC (SEQ ID NO: 77) or VEPKSC (SEQ ID NO: 78) or AEPKSC (SEQ ID NO: 79) on one polypeptide chain and GFNRGEC (SEQ ID NO: 80) or FNRGEC (SEQ ID NO: 80) on the other polypeptide chain. SEQ ID NO:81) (see US Pat. No. 9,296,816).

In a preferred embodiment, the heterodimer-promoting domain is a tandem repeat coil domain of opposite charge, e.g., an "E-coil" helical domain (SEQ ID NO: 82) whose glutamate residues form a negative charge at pH 7: E VAAL E K- E VAAL E K- E VAAL E K- E VAAL E K), and a "K-coil" domain whose lysine residues form a positive charge at pH 7 (SEQ ID NO: 83): K VAAL K E- K VAAL K E- K VAAL K E- K VAAL K E). The presence of such charged domains facilitates binding between the first and second polypeptides, thus facilitating heterodimer formation. Heterodimer-promoting domains can be utilized that include modifications of the E-coil and K-coil sequences to include one or more cysteine residues. The presence of such cysteine residues allows a coil present on one polypeptide chain to become covalently linked to a complementary coil present on another polypeptide chain, thereby covalently linking the polypeptide chains to each other. and increase the stability of the diabody. Examples of such particularly preferred heterodimer-promoting domains include a modified E-coil having the amino acid sequence E VAA CE K- E VAAL E K- E VAAL E K- E VAAL E K (SEQ ID NO: 84) and the amino acid sequence K and a modified K coil with VAA CK EK VAAL KE K VAAL KE VAAL KE (SEQ ID NO: 85).

To improve the in vivo pharmacokinetic properties of the diabody, as disclosed in PCT Publication No. WO 2012/018687, the diabody may contain a polypeptide portion of a serum binding protein at one or more termini of the diabody. can be modified to include Most preferably, such polypeptide portion of the serum binding protein is placed at the C-terminus of the polypeptide chain of the diabody. Albumin is the most abundant protein in plasma and has a half-life of 19 days in humans. Albumin has several small-molecule binding sites that allow non-covalent binding to other proteins, thereby prolonging their serum half-life. Albumin-binding domain 3 (ABD3) of protein G of Streptococcus strain G148 is composed of 46 amino acid residues that form a stable triple-helical bundle and has broad albumin-binding specificity (Johansson, M.U. et al. (2002). ) "Structure, Specificity, And Mode Of Interaction For Bacterial Albumin-Binding Modules," J. Biol. Chem. 277(10):8114-8120). Therefore, a particularly preferred polypeptide portion of the serum binding protein for improving the in vivo pharmacokinetic properties of the diabody is the albumin binding domain (ABD) from Streptococcus protein G, more preferably protein G of Streptococcus strain G148. albumin binding domain 3 (ABD3) (SEQ ID NO: 86): LAEAKVLANR ELDKYGVSDY YKNLIDNAKS AEGVKALIDE ILAALP.

As disclosed in PCT Publication No. WO 2012/162068 (incorporated herein by reference), "deimmunized" variants of SEQ ID NO:86 attenuate or have the ability to remove Based on combinatorial mutagenesis results, the following combinations of substitutions are believed to be preferred substitutions for forming such deimmunized ABDs: 66D/70S+71A; 66S/70S+71A; 66S/70S+79A; 64A/65A/71A. 64A/65A/71A+66S; 64A/65A/71A+66D; 64A/65A/71A+66E; 64A/65A/79A+66S; 64A/65A/79A+66D; Mutant ABD with modifications L64A, I65A and D79A or modifications N66S, T70S and D79A. Since such deimmunized ABD exhibits substantially wild-type binding while providing attenuated MHC class II binding, the amino acid sequence:
LAEAKVLANR ELDKYGVSDY _{YKNLI D66} NAK S70 A71 _EGVKALIDE ILAALP (SEQ ID NO: 87),
or amino acid sequence:
_LAEAKVLANR ELDKYGVSDY YKN A64 A65 _{NNAKT VEGVKALI} A79 E ILAALP (SEQ ID NO: 88),
or amino acid sequence:
LAEAKVLANR ELDKYGVSDY _YKNLI S66 NAK S70 _VEGVKALI A79 E _ILAALP (SEQ ID NO: 89)
Particularly preferred is a mutant deimmunized ABD with Thus, the first polypeptide chain of such diabodies having an ABD preferably comprises a third linker (linker 3 ) and intervenes between the E-coil (or K-coil) domain and the ABD (which is preferably deimmunized ABD). A preferred sequence for such linker 3 is GGGS (SEQ ID NO:71).
C. Multispecific diabodies containing Fc regions

One embodiment of the present invention relates to multispecific diabodies capable of simultaneously binding an ADAM9 epitope comprising an Fc region and a second epitope (i.e., different ADAM9 or non-ADAM9 epitopes of a molecule). The addition of IgG CH2-CH3 domains to one or both of the diabody polypeptide chains such that complexation of the diabody chains results in the formation of the Fc region increases biological half-life and/or Or the valence of the diabody changes. Such diabodies, by virtue of their sequence, allow the polypeptide chains to covalently link to each other to form a covalently linked diabody capable of simultaneously binding an epitope of ADAM9 and a second epitope. Contains two or more polypeptide chains. Incorporation of IgG CH2-CH3 domains into both diabody polypeptides allows the formation of a two-chain bispecific Fc region-containing diabody (Figure 2).

Alternatively, incorporation of IgG CH2-CH3 domains onto only one of the diabody polypeptides allows the formation of more complex four-chain bispecific Fc region-containing diabodies. (Figures 3A-3C). FIG. 3C shows a representative four-chain diabody with a constant light (CL) domain and a constant heavy CH1 domain, although fragments of such domains as well as other polypeptides can alternatively be used (e.g. , Figures 3A and 3B, US Patent Publication Nos. 2013-0295121; 2010-0174053 and 2009-0060910; European Patent Publication No. EP2714079; EP2601216; 2012/162068; WO2012/018687; WO2010/080538). Thus, for example, instead of the CH1 domain, a peptide having the amino acid sequence GVEPKSC (SEQ ID NO: 77), VEPKSC (SEQ ID NO: 78), or AEPKSC (SEQ ID NO: 79) from the hinge region of human IgG can be used, and CL Instead of domains, the C-terminal 6 amino acids of the human kappa light chain, GFNRGEC (SEQ ID NO:80) or FNRGEC (SEQ ID NO:81) can be used. A representative peptide containing four-chain diabody is shown in FIG. 3A. Alternatively or additionally, an "E-coil" helix domain (SEQ ID NO:82): E VAAL E K- E VAAL E K- E VAAL E K- E VAAL E K or SEQ ID NO:84): E VAA CE K- E VAAL E K- E VAAL E K- E VAAL E K); and the "K coil" domain (SEQ ID NO:83): K VAAL K E- K VAAL K E- K VAAL K E- K VAAL K E or SEQ ID NO:85 ): K VAA CK EK VAAL KE- K VAAL KE- K VAAL KE ) can be used. A representative coil domain containing four-chain diabody is shown in FIG. 3B.

The Fc Region-containing molecules of the invention may comprise additional intervening spacer peptides (linkers), generally such linkers are between the heterodimer-promoting domain (e.g., E-coil or K-coil) and the CH2-CH3 domains and /or incorporated between the CH2-CH3 domain and the variable domain (ie, VH or VL). Typically, the additional linker comprises 3-20 amino acid residues and may optionally comprise all or part of the IgG hinge region (preferably the cysteine-containing portion of the IgG hinge region). Linkers that can be used in the bispecific Fc Region-containing diabody molecules of the invention include: GGGS (SEQ ID NO:71), LGGGSG (SEQ ID NO:72), GGGSGGGSGGG (SEQ ID NO:73), ASTKG (SEQ ID NO:74), LEPKSS (SEQ ID NO: 75), APSSS (SEQ ID NO: 76), APSSSPME (SEQ ID NO: 90), VEPKSADKTHTCPCP (SEQ ID NO: 91), LEPKSADKTHTCPPC (SEQ ID NO: 92), DKTHTCPPCP (SEQ ID NO: 93), GGC, and GGG. LEPKSS (SEQ ID NO: 75) may be used in place of GGG or GGC to facilitate cloning. Additionally, amino acids GGG, or LEPKSS (SEQ ID NO:75) may be immediately followed by DKTHTCPPCP (SEQ ID NO:93) to form another linker: GGGDKTHTCPPCP (SEQ ID NO:94); and LEPKSSDKTHTCPPCP (SEQ ID NO:95). A bispecific Fc region-containing molecule of the invention may incorporate an IgG hinge region in addition to or instead of a linker. Exemplary hinge regions include: EPKSCDKTHTCPPCP from IgG1 (SEQ ID NO:96), ERKCCVECPPCP from IgG2 (SEQ ID NO:97), ELKTPLGDTT HTCPRCPEPK SCDTPPPCPR CPEPKSCDTP PPCPRCPEPK SCDTPPPCPR CP from IgG3 (SEQ ID NO:206), IgG4. ESKYGPPCPSCP (SEQ ID NO: 98), and ESKYGPPCP CP (SEQ ID NO: 99) from IgG4, which contains a stabilizing S228P substitution (underlined) (numbered by the EU index as described in Kabat) to reduce strand exchange. Hinge mutant.

As presented in Figures 3A-3C, the Fc Region-containing diabodies of the invention can comprise four chains. The first and third polypeptide chains of such diabodies are composed of three domains: (i) a VL1-containing domain, (ii) a VH2-containing domain, (iii) a heterodimer-promoting domain, and (iv) a CH2-CH3 sequence. Contains domains that contain . The second and fourth polypeptide chains comprise: (i) a VL2-containing domain, (ii) a VH1-containing domain, and (iii) a heterodimer-promoting domain, wherein the heterodimer-promoting domain is the first/third polypeptide chain and promote dimerization of the second/fourth polypeptide chain. The VL and/or VH domains of the third and fourth polypeptide chains and the VL and/or VH domains of the first and second polypeptide chains may be the same or different, monospecific, bivalent Allows tetravalent binding that is either bispecific or tetraspecific. The notations "VL3" and "VH3" refer to the light and heavy chain variable domains, respectively, that bind the "tertiary" epitope of such diabodies. Similarly, the notations "VL4" and "VH4" refer to the light and heavy chain variable domains, respectively, that bind the "fourth" epitope of such diabodies. The general structures of the polypeptide chains of representative four-chain bispecific Fc Region-containing diabodies of the invention are presented in Table 2:

ＨＰＤ＝ヘテロダイマー促進ドメイン

HPD = heterodimer promoting domain

In a specific embodiment, the diabodies of the invention are bispecific, tetravalent (i.e., with four epitope binding sites), Fc-containing diabodies composed of four total polypeptide chains (Fig. 3A-3C). The bispecific, tetravalent, Fc-containing diabodies of the invention have two epitope binding sites immunospecific for ADAM9, which may be capable of binding to the same epitope of ADAM9 or to different epitopes of ADAM9. , comprising two epitope binding sites that are immunospecific for a second molecule, which may be capable of binding the same epitope of the second molecule or different epitopes of the second molecule. Preferably, the second molecule is a molecule present on the surface of effector cells such as T lymphocytes, natural killer (NK) cells or other monocytes (e.g. CD2, CD3, CD8, CD16, T cell receptor (TCR), NKG2D, etc.).

In further embodiments, the Fc Region-containing diabodies of the invention may comprise three polypeptide chains. The first polypeptide of such a diabody comprises three domains: (i) a VL1-containing domain, (ii) a VH2-containing domain and (iii) a domain containing a CH2-CH3 sequence. The second polypeptide of such a diabody is: (i) a VL2-containing domain, (ii) a VH1-containing domain and (iii) promoting heterodimerization and covalent association with the first polypeptide chain of the diabody. Contains domains. A third polypeptide of such a diabody comprises a CH2-CH3 sequence. Thus, the first and second polypeptide chains of such a diabody are joined together to provide a VL1/VH1 epitope binding site capable of binding a first antigen (i.e., ADAM9 or a molecule containing a second epitope). either), and a VL2/VH2 epitope binding site capable of binding a second antigen (ie either the molecule containing the second epitope or ADAM9). The first and second polypeptides are linked to each other through disulfide bonds containing cysteine residues in their respective third domains. In particular, the first and third polypeptide chains are conjugated to each other to form an Fc region that is stabilized through disulfide bonds. Such bispecific diabodies have enhanced potency. Figures 4A and 4B show the structure of such a diabody. Such Fc region-containing diabodies can have either of two orientations (Table 3):

ＨＰＤ＝ヘテロダイマー促進ドメイン

HPD = heterodimer promoting domain

In a specific embodiment, the diabodies of the invention are bispecific, bivalent (i.e., having two epitope binding sites), Fc-containing diabodies composed of a total of three polypeptide chains ( 4A-4B). The bispecific, bivalent Fc-containing diabodies of the invention comprise one epitope binding site immunospecific for ADAM9 and one epitope binding site immunospecific for a second molecule. Preferably, the second molecule is a molecule present on the surface of effector cells such as T lymphocytes, natural killer (NK) cells or other monocytes (e.g. CD2, CD3, CD8, CD16, T cell receptor (TCR), NKG2D, etc.).

In a further embodiment, an Fc region-containing diabodies may comprise a total of 5 polypeptide chains. In certain embodiments, two of said five polypeptide chains have the same amino acid sequence. The first polypeptide chain of such diabodies comprises: (i) a VH1-containing domain, (ii) a CH1-containing domain, and (iii) a domain comprising a CH2-CH3 sequence. The first polypeptide chain can be the heavy chain of an antibody comprising VH1 and the heavy chain constant region. The second and fifth polypeptide chains of such diabodies comprise: (i) a VL1-containing domain and (ii) a CL-containing domain. The second and/or fifth polypeptide chain of such diabodies can be the light chain of an antibody comprising VL1 complementary to the VH1 of the first/third polypeptide chain. The first, second and/or fifth polypeptide chains can be isolated from naturally occurring antibodies. Alternatively, they can be recombinantly constructed. The third polypeptide chain of such a diabody is: (i) a VH1-containing domain, (ii) a CH1-containing domain, (iii) a domain comprising a CH2-CH3 sequence, (iv) a VL2-containing domain, (v) a VH3-containing domain; and (vi) a heterodimer-promoting domain, wherein the heterodimer-promoting domain promotes dimerization of the third and fourth chains. The fourth polypeptide of such a diabody undergoes: (i) a VL3-containing domain, (ii) a VH2-containing domain, and (iii) heterodimerization and covalent linkage to the third polypeptide chain of the diabody. and promoting domains.

Thus, the first and second and third and fifth polypeptide chains of such a diabody join together to form two VL1/VH1 epitope binding sites capable of binding the first epitope. do. The third and fourth polypeptide chains of such diabodies are joined together to form a VL2/VH2 epitope binding site capable of binding a second epitope, and a third epitope capable of binding Forms a VL3/VH3 binding site. The first and third polypeptides are linked together by disulfide bonds containing cysteine residues in their respective constant regions. In particular, the first and third polypeptide chains are conjugated to each other to form the Fc region. Such multispecific diabodies have enhanced potency. FIG. 5 shows the structure of such a diabody. It is understood that the VL1/VH1, VL2/VH2, and VL3/VH3 domains may be the same or different, allowing binding that is monospecific, bispecific or trispecific. As presented herein, these domains are preferably selected to bind an epitope of ADAM9, a second molecular epitope and optionally a third molecular epitope.

The VL and VH domains of the polypeptide chain are selected to form a VL/VH binding site specific for the desired epitope. The VL/VH binding sites formed by the association of the polypeptide chains can be identical or different to allow tetravalent binding that can be monospecific, bispecific, trispecific or tetraspecific. may In particular, the VL and VH domains are such that the multivalent diabody has two binding sites for the first epitope and two binding sites for the second epitope, also three binding sites for the first epitope and one binding site for the second epitope, Alternatively, one can choose to include two binding sites for the first epitope, one binding site for the second epitope and one binding site for the third epitope (as shown in Figure 5). The general structures of the polypeptide chains of representative five-chain Fc region-containing diabodies of the invention are presented in Table 4:

ＨＰＤ＝ヘテロダイマー促進ドメイン

HPD = heterodimer promoting domain

In a specific embodiment, the diabodies of the invention comprise two epitope binding sites immunospecific for ADAM9 (which may be capable of binding the same epitope of ADAM9 or different epitopes of ADAM9) and bispecific, composed of a total of five polypeptide chains having two epitope binding sites specific for a second molecule, which may be capable of binding to the same epitope of the second molecule or to different epitopes of the second molecule; Tetravalent (ie, having four binding sites) Fc-containing diabodies. In another embodiment, the bispecific tetravalent Fc-containing diabodies of the invention have three epitope binding sites immunospecific for ADAM9 (same epitope of ADAM9 or two or three different epitopes of ADAM9). bindable) and one epitope binding site specific for the second molecule. In another embodiment, the bispecific tetravalent Fc-containing diabodies of the invention have one epitope binding site immunospecific for ADAM9 and three epitope binding sites specific for a second molecule (second molecule can bind to the same epitope of or two or three different epitopes of the second molecule). As indicated above, the VL and VH domains can be selected to allow trispecific binding. Accordingly, the present invention may also include trispecific, tetravalent Fc-containing diabodies. The trispecific tetravalent Fc-containing diabodies of the invention have two epitope binding sites immunospecific for ADAM9, one epitope binding site immunospecific for a second molecule, and an epitope binding site immunospecific for a third molecule. and one epitope binding site. In certain embodiments, the second molecule is a molecule present on the surface of effector cells such as T lymphocytes, natural killer (NK) cells or other mononuclear cells (e.g., CD2, CD3, CD8, CD16, T cell receptor (TCR), NKG2D, etc.). In certain embodiments, the second molecule is CD3 and the third molecule is CD8.
D. Trivalent binding molecules comprising an Fc region

A further embodiment of the invention relates to a trivalent binding molecule comprising an Fc region capable of simultaneously binding a first epitope, a second epitope and a third epitope, wherein at least one such epitope is not identical to each other. Such trivalent binding molecules contain three epitope binding sites, two of which are diabody-type binding domains, which provide binding site A and binding site B, one of which is Fab-type binding. domain, or scFv-type binding domain, which provides binding site C (see, eg, FIGS. 6A-6F and PCT Publication Nos: WO2015/184207; and WO2015/184203). Such trivalent binding molecules therefore have a "VL1"/"VH1" domain capable of binding a first epitope, a "VL2"/"VH2" domain capable of binding a second epitope, and and "VL3" and "VH3" domains capable of binding a "third" epitope of such a trivalent binding molecule. A "diabody-type binding domain," as described above, is a type of epitope binding site present in diabodies, particularly DART® diabodies. Each "Fab-type binding domain" and "scFv-type binding domain" is an epitope binding site formed by the interaction of the VL domain of an immunoglobulin light chain and the complementary VH domain of an immunoglobulin heavy chain. Fab-type binding domains are those in which the two polypeptide chains forming the Fab-type binding domain contain only a single epitope binding site, whereas the two polypeptide chains forming the diabody-type binding domain contain at least two epitope binding sites. It differs from the diabody-type binding domain in that it contains an epitope binding site. Similarly, scFv-type binding domains also differ from diabody-type binding domains in that they contain only a single epitope binding site. Thus, as used herein, Fab-type and scFv-type binding domains are distinct from diabody-type binding domains.

Although typically a trivalent binding molecule of the invention comprises four different polypeptide chains (see FIGS. 6A-6B), the molecule may, for example, link such polypeptide chains (eg, via peptide bonds) ) by fusing together, or by splitting such polypeptide chains to form additional polypeptide chains, or by joining fewer or additional polypeptide chains through disulfide bonds. It may contain fewer or more polypeptide chains. Figures 6C-6F illustrate this aspect of the invention by schematically drawing such a molecule having three polypeptide chains. As presented in Figures 6A-6F, the trivalent binding molecules of the invention have a diabody-type binding domain that is N-terminal (Figures 6A, 6C and 6D) or C-terminal (Figures 6B, 6E and 6F) to the Fc region. ).

In certain embodiments, the first polypeptide chain of such trivalent binding molecules of the invention are: (i) a VL1-containing domain, (ii) a VH2-containing domain, (iii) a heterodimer-promoting domain, and (iv) ) contains a domain containing the CH2-CH3 sequence. The VL1 and VL2 domains are located N-terminally or C-terminally to the CH2-CH3 containing domains as presented in Table 4 (see also Figures 6A and 6B). The second polypeptide chain of such embodiments comprises: (i) a VL2-containing domain, (ii) a VH1-containing domain, and (iii) a heterodimer-promoting domain. The third polypeptide chain of such embodiments comprises: (i) a VH3-containing domain, (ii) a CH1-containing domain and (iii) a domain comprising a CH2-CH3 sequence. The third polypeptide chain may be the heavy chain of an antibody comprising VH3 and the heavy chain constant region, or a polypeptide comprising such domains. The fourth polypeptide of such embodiments comprises: (i) a VL3-containing domain and (ii) a CL-containing domain. The fourth polypeptide chain may be the light chain of an antibody containing VL3 that is complementary to VH3 of the third polypeptide chain, or a polypeptide containing such a domain. A third or fourth polypeptide chain can be isolated from a naturally occurring antibody. Alternatively, they may be constructed recombinantly, synthetically, or by other means.

the light chain variable domains of the first and second polypeptide chains are joined together by their VL1/VH2 (or their VL2/VH1) domains and are capable of binding either the first or second epitope It is separated from the heavy chain variable domain of such a polypeptide chain by an intervening spacer peptide that is too short in length to allow the site to form. A preferred intervening spacer peptide (linker 1) for this purpose has the sequence (SEQ ID NO: 69): GGGSGGGG. The other domains of the trivalent binding molecule may be separated by one or more intervening spacer peptides (linkers), which may optionally contain cysteine residues. In particular, as noted above, such linkers are typically between variable domains (i.e., VH or VL) and peptide heterodimer-promoting domains (e.g., E-coil or K-coil) and such Incorporated between the peptide heterodimer-promoting domain (eg, E-coil or K-coil) and the CH2-CH3 domain. Exemplary linkers useful for generating trivalent binding molecules are presented above and also in PCT Application Nos: PCT/US15/33081; and PCT/US15/33076. Thus, the first and second polypeptide chains of such trivalent binding molecules are joined together to form a VL1/VH1 binding site capable of binding a first epitope and a VL2/VH2 binding site capable of binding a second epitope. Forms binding sites. The third and fourth polypeptide chains of such trivalent binding molecules are joined together to form a VL3/VH3 binding site capable of binding a third epitope.

As noted above, a trivalent binding molecule of the invention may comprise three polypeptides. A trivalent binding molecule comprising three polypeptide chains can optionally be obtained by linking the N-terminal domain of a fourth polypeptide with the VH3-containing domain of a third polypeptide (e.g. an intervening spacer peptide (using linker 4)). Alternatively, the third polypeptide chain of the trivalent binding molecule of the invention comprises the following domains: (i) a VL3-containing domain, (ii) a VH3-containing domain, and (iii) a domain comprising a CH2-CH3 sequence utilized, wherein VL3 and VH3 are separated from each other by an intervening spacer peptide long enough (at least 9 amino acid residues or more) to join these domains and form an epitope binding site. One preferred intervening spacer peptide for this purpose has the sequence: GGGGSGGGGSGGGGGS (SEQ ID NO: 100).

The VL1/VH1, VL2/VH2 and VL3/VH3 domains of such trivalent binding molecules may be different to allow binding that is monospecific, bispecific or trispecific. is understood. In particular, the VL and VH domains are such that the trivalent binding molecule comprises two binding sites for the first epitope and one binding site for the second epitope, or one binding site for the first epitope and a second Choose to contain two binding sites for the epitope or to contain one binding site for the first epitope, one binding site for the second epitope and one binding site for the third epitope. be able to.

However, as presented herein, these domains are preferably selected to bind an epitope of ADAM9, a second molecular epitope, and a third molecular epitope. In certain embodiments, the second molecule is a molecule present on the surface of effector cells such as T lymphocytes, natural killer (NK) cells or other mononuclear cells (e.g., CD2, CD3, CD8, CD16, T cell receptor (TCR), NKG2D, etc.). In certain embodiments, the third molecule is CD8.

The general structures of the polypeptide chains of trivalent binding molecules of the invention are presented in FIGS. 6A-6F and Table 5:

ＨＰＤ＝ヘテロダイマー促進ドメイン

HPD = heterodimer promoting domain

One embodiment of the invention relates to a trivalent binding molecule comprising two epitope binding sites of ADAM9 and one epitope binding site of a second molecule. The two epitope binding sites of ADAM9 can bind the same epitope or different epitopes. Another embodiment of the invention relates to a trivalent binding molecule comprising one epitope binding site of ADAM9 and two epitope binding sites of a second molecule. The two epitope binding sites on the second molecule can bind the same epitope or different epitopes on the second molecule. A further embodiment of the invention relates to a trispecific trivalent binding molecule comprising one epitope binding site of ADAM9, one epitope binding site of the second molecule and one epitope binding site of the third molecule. In certain embodiments, the second molecule is a molecule present on the surface of effector cells such as T lymphocytes, natural killer (NK) cells or other mononuclear cells (e.g., CD2, CD3, CD8, CD16, T cell receptor (TCR), NKG2D, etc.). In certain embodiments, the second molecule is CD3 and the third molecule is CD8. As indicated above, such trivalent binding molecules may comprise 3, 4, 5, or more polypeptide chains.
IX. Constant domains and variant Fc regions

Provided herein are antibody "constant domains" useful in generating ADAM9 binding molecules (eg, antibodies, diabodies, trivalent binding molecules, etc.) of the invention.

A preferred CL domain is a human IgG CLK domain. The amino acid sequence of an exemplary human CLK domain is (SEQ ID NO: 101):
RTVAAPSVFI FPPSDEQLKS GTASVVCLLN NFYPREAKVQ WKVDNALQSG NSQESVTEQD SKDSTYSLSS
TLTLSKADYE KHKVYACEVT HQGLSSPVTK SFNRGEC
is.

Alternatively, an exemplary CL domain is a human IgG CLλ domain. The amino acid sequence of an exemplary human CLλ domain is (SEQ ID NO: 102):
QPKAAPSVTL FPPSSEELQA NKATLVCLIS DFYPGAVTVA WKADSSPVKA GVETTPSKQS NNKYAASSYL
SLTPEQWKSH RSYSCQVTHE GSTVEKTVAP TECS
is.

As provided herein, ADAM9 binding molecules of the invention can include an Fc region. The Fc region of such molecules of the invention can be of any isotype (eg, IgG1, IgG2, IgG3, or IgG4). ADAM9-binding molecules of the invention may further comprise a CH1 domain and/or hinge region. If present, the CH1 domain and/or hinge region may be of any isotype (eg, IgG1, IgG2, IgG3, or IgG4), preferably of the same isotype as the desired Fc region.

An exemplary CH1 domain is a human IgG1 CH1 domain. The amino acid sequence of an exemplary human IgG1 CH1 domain is (SEQ ID NO: 103):
ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT
VPSSSLGTQT YICNVNHKPS NTKVDKRV
is.

An exemplary CH1 domain is a human IgG2 CH1 domain. The amino acid sequence of an exemplary human IgG2 CH1 domain is (SEQ ID NO: 104):
ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT
VPSSNFGTQT YTCNVDHKPS NTKVDKTV
is.

An exemplary CH1 domain is a human IgG3 CH1 domain. The amino acid sequence of an exemplary human IgG3 CH1 domain is (SEQ ID NO:207):
ASTKGPSVFP LAPCSRSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT
VPSSSLGTQT YTCNVNHKPS NTKVDKRV
is.

An exemplary CH1 domain is a human IgG4 CH1 domain. The amino acid sequence of an exemplary human IgG4 CH1 domain is (SEQ ID NO: 105):
ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT
VPSSSLGTKT YTCNVDHKPS NTKVDKRV
is.

One exemplary hinge region is the human IgG1 hinge region. An exemplary human IgG1 hinge region amino acid sequence is (SEQ ID NO:96): EPKSCDKTHTCPPCP.

Another exemplary hinge region is the human IgG2 hinge region. An exemplary human IgG2 hinge region amino acid sequence is (SEQ ID NO:97): ERKCCVECPPCP.

Another exemplary hinge region is the human IgG4 hinge region. The amino acid sequence of an exemplary human IgG4 hinge region is (SEQ ID NO:98): ESKYGPPCPSCP. As noted above, the IgG4 hinge region may contain stabilizing mutations such as the S228P substitution. The amino acid sequence of an exemplary stabilizing IgG4 hinge region is (SEQ ID NO:99): ESKYGPPCPPCP.

The Fc Region of the Fc Region-containing molecules (e.g., antibodies, diabodies, trivalent binding molecules, etc.) of the invention can be either a complete Fc Region (e.g., a complete IgG Fc Region) or only a fragment of an Fc Region. good. Optionally, the Fc region of the Fc region-containing molecules of the invention lacks a C-terminal lysine amino acid residue.

In traditional immune function, the interaction of antibody-antigen complexes with cells of the immune system results in a transition from effector functions such as antibody-dependent cytotoxicity, mast cell degranulation, and phagocytosis to lymphocyte proliferation and antibody secretion. A wide range of responses is obtained, extending to immunoregulatory signals such as regulation. All of these interactions are initiated by the binding of the Fc region of antibodies or immune complexes to specialized cell surface receptors on hematopoietic cells. Various cellular responses triggered by antibodies and immune complexes are due to the structural heterogeneity of three Fc receptors: FcγRI (CD64), FcγRII (CD32), and FcγRIII (CD16). FcγRI (CD64), FcγRIIA (CD32A) and FcγRIII (CD16) are activating (ie, immune system enhancing) receptors; FcγRIIB (CD32B) is an inhibitory (ie, immune system suppressing) receptor. Furthermore, interaction with neonatal Fc receptors (FcRn) mediates recycling of IgG molecules from endosomes to the cell surface and release into the blood. The amino acid sequences of exemplary wild-type IgGl (SEQ ID NO: 1), IgG2 (SEQ ID NO: 2), IgG3 (SEQ ID NO: 3), and IgG4 (SEQ ID NO: 4) are provided above.

Modifications of the Fc region may result in altered phenotypes, such as altered serum half-life, altered stability, altered sensitivity to cellular enzymes or altered effector function. Accordingly, it may be desirable to modify the Fc region-containing ADAM9 binding molecules of the invention with respect to effector function, eg, to enhance the efficacy of such molecules in treating cancer. Attenuation or elimination of effector function is desirable in some cases, for example, antibodies whose mechanism of action involves blocking or antagonism, but not killing of cells bearing the target antigen. Increased effector function is generally desirable when directed to unwanted cells, e.g., tumors and heterogeneous cells where FcγRs are expressed at low levels, e.g., tumor-specific B cells with low levels of FcγRIIB (e.g. , non-Hodgkin's lymphoma, CLL, and Burkitt's lymphoma). Molecules of the invention having such conferred or altered effector function activity are useful in the treatment and/or prevention of diseases, disorders or infections in which enhanced efficacy of effector function activity is desired.

Thus, in certain embodiments, the Fc region of an Fc region-containing molecule of the invention can be an engineered variant Fc region. The Fc region of the bispecific Fc region-containing molecules of the invention may have the ability to bind one or more Fc receptors (e.g., FcγR), more preferably such variant Fc regions are FcγRIA (CD64 ), FcγRIIA (CD32A), FcγRIIB (CD32B), FcγRIIIA (CD16a) or FcγRIIIB (CD16b) (relative to the binding exhibited by the wild-type Fc region), e.g., activating receptors and/or substantially reduced or no ability to bind to inhibitory receptor(s). Thus, the Fc region of an Fc Region-containing molecule of the invention may comprise part or all of the CH2 domain and/or part or all of the CH3 domain of a complete Fc region, or alternatively mutated CH2 and/or mutated CH3 sequences ( For example, which may contain one or more insertions and/or one or more deletions with respect to the CH2 or CH3 domains of the complete Fc region). Such Fc regions may comprise non-Fc polypeptide portions, or may comprise portions of non-naturally occurring complete Fc regions, or non-naturally occurring orientations of the CH2 and/or CH3 domains (e.g., two CH2 domain or two CH3 domains, or a CH3 domain linked to a CH2 domain in the N-terminal to C-terminal direction).

Fc regions that have been identified as altering effector function, including modifications that increase binding to activating receptors such as FcγRIIA (CD16A) and decrease binding to inhibitory receptors such as FcγRIIB (CD32B) Modifications are known in the art (e.g. Stavenhagen, J.B. et al. (2007) “Fc Optimization Of Therapeutic Antibodies Enhances Their Ability To Kill Tumor Cells In Vitro And Controls Tumor Expansion In Vivo Via Low-Affinity Activating Fcgamma Receptors, 57(18):8882-8890). Table 6 lists exemplary modifications that increase binding to activating receptors and/or decrease binding to inhibitory receptors. Double, triple, quadruple and quintuple substitutions are listed (numbering is the EU index as in Kabat, substitutions relative to the amino acid sequence of SEQ ID NO:1).

Exemplary variants of a human IgG1 Fc region with reduced binding to CD32B and/or increased binding to CD16A comprise F243L, R292P, Y300L, V305I or P396L substitutions, where the numbering is EU as in Kabat. exponential. These amino acid substitutions can be present in the human IgG1 Fc region in any combination. In one embodiment, the mutated human IgG1 Fc region comprises F243L, R292P and Y300L substitutions. In another embodiment, the mutated human IgG1 Fc region comprises F243L, R292P, Y300L, V305I and P396L substitutions.

In certain embodiments, the Fc region of an ADAM9 binding molecule of the invention exhibits reduced binding to FcγRIA (CD64), FcγRIIA (CD32A), FcγRIIB (CD32B), FcγRIIIA (CD16a) or FcγRIIIB (CD16b) (or substantially no) is preferred (relative to the binding exhibited by the wild-type IgG1 Fc region (SEQ ID NO: 1)). In a specific embodiment, the ADAM9 binding molecules of the invention comprise an IgG Fc region that exhibits reduced ADCC effector function. In preferred embodiments, the CH2-CH3 domain of such ADAM9 binding molecules comprises any 1, 2, 3, or 4 of the substitutions: L234A, L235A, D265A, N297Q, and N297G, wherein the numbering is of the EU index as in Kabat. In another embodiment, the CH2-CH3 domain comprises the N297Q, N297G, L234A and L235A or D265A substitutions as these mutations abolish FcR binding. Alternatively, substantially reduced (or substantially no) binding and/or reduced binding to FcγRIIIA (CD16a) (compared to the binding and effector functions exhibited by the wild-type IgG1 Fc region (SEQ ID NO: 1)) It utilizes the CH2-CH3 domains of naturally occurring Fc regions that exhibit effector function. In specific embodiments, an ADAM9 binding molecule of the invention comprises an IgG2Fc region (SEQ ID NO:2) or an IgG4Fc region (SEQ ID NO:4). When utilizing an IgG4 Fc region, the invention also includes the introduction of stabilizing mutations such as the aforementioned hinge region S228P substitution (see, eg, SEQ ID NO:99). Since the N297G, N297Q, L234A, L235A and D265A substitutions abolish effector function, these substitutions are preferably not used in situations where effector function is desired.

A preferred IgG1 sequence for the CH2 and CH3 domains of an Fc Region-containing molecule of the invention with reduced or abolished effector function comprises the substitution L234A/L235A (underlined) (SEQ ID NO: 106):
APE AA GGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY
RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVK
GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS
LSLSPG X
where X is lysine (K) or absent.

A second preferred IgG1 sequence for the CH2 and CH3 domains of the Fc Region-containing molecules of the invention contains the S442C substitution (shown underlined), thus allowing the two CH3 domains to covalently bind to each other via a disulfide bond. or allow conjugation of drug moieties. The amino acid sequence of such a molecule is (SEQ ID NO: 107):
APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY
RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVK
GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS
L C LSPG X
where X is lysine (K) or absent.

A third preferred IgG1 sequence for the CH2 and CH3 domains of the Fc Region-containing molecules of the invention is a disulfide bond or conjugation of the drug moiety with the L234A/L235A substitution (underlined) that reduces or eliminates effector function. S442C substitution (underlined) that allows them to be covalently linked to each other by gation. The amino acid sequence of such a molecule is (SEQ ID NO: 108):
APE AA GGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY
RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVK
GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS
L C LSPG X
and X is lysine (K) or absent.

The serum half-life of Fc region-containing proteins can be increased by increasing the binding affinity of the Fc region of FcRn. The term "half-life," as used herein, refers to a pharmacokinetic property of molecules that is a measure of the average survival time of molecules after their administration. Half-life is known from the subject's (e.g., human patient or other mammal) body or particular compartment thereof, e.g., as measured in serum, i.e., circulating half-life, or in other tissues. It can be expressed as the time required to remove fifty percent (50%) of the mass of molecules. In general, increased half-life results in increased mean residence time (MRT) in circulation of an administered molecule.

In some embodiments, an ADAM9-binding molecule of the invention comprises a variant Fc region comprising at least one amino acid modification relative to a wild-type Fc region, such that said molecule is (compared to a molecule comprising a wild-type Fc region ) and have a half-life. In some embodiments, the ADAM9-binding molecule of the invention comprises a variant IgG Fc region, wherein said variant Fc region is from the group consisting of 303, 305, 307, 308, 309, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424, 428, 433, 434, 435, and 436 Include half-life extending amino acid substitutions at one or more selected positions, where the numbering is of the EU index as in Kabat. Many mutations are known in the art that can increase the half-life of Fc region-containing molecules, including M252Y, S254T, T256E, and combinations thereof. For example, US Pat. Nos. 6,277,375, 7,083,784; 7,217,797, 8,088,376, which are incorporated herein by reference in their entirety. U.S. Publication No. 2002/0147311; U.S. Publication No. 2007/0148164; and PCT Publication No. WO 98/23289; See mutation. ADAM9-binding molecules with increased half-lives also have Also included are those with variant Fc regions that contain more than one substitution. In particular two or more substitutions selected from T250Q, M252Y, S254T, T256E, K288D, T307Q, V308P, A378V, M428L, N434A, H435K and Y436I, the numbering being of the EU index as in Kabat.

In a specific embodiment, the ADAM9-binding molecules of the invention have the substitution:
(A) M252Y, S254T and T256E;
(B) M252Y and S254T;
(C) M252Y and T256E;
(D) T250Q and M428L;
(E) T307Q and N434A;
(F) A378V and N434A;
(G) N434A and Y436I;
(H) V308P and N434A; or (I) K288D and H435K
has a variant IgG Fc region comprising

In a preferred embodiment, the ADAM9 binding molecule of the invention has a variant IgG Fc region comprising any one, two or three of the substitutions: M252Y, S254T and T256E. The invention further:
ADAM9 binding molecules with variant Fc regions comprising (A) one or more mutations that alter effector function and/or FcγR; and (B) one or more mutations that increase serum half-life are encompassed.

A fourth preferred IgG1 sequence for the CH2 and CH3 domains of the Fc Region-containing molecules of the invention contains M252Y, S254T and T256E substitutions (underlined) to extend serum half-life. The amino acid sequence of such molecule (SEQ ID NO:200) is:
APELLGGPSV FLFPPKPKDT L Y I T R E PEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY
RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVK
GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS
LSLSPG X
where X is lysine (K) or absent.

A fifth preferred IgG1 sequence for the CH2 and CH3 domains of the Fc Region-containing molecules of the invention is the L234A/L235A substitution (underlined) that reduces or eliminates effector function, and the M252Y sequence that extends serum half-life. , S254T and T256E substitutions (underlined). The amino acid sequence of such molecule (SEQ ID NO:201) is:
APE AA GGPSV FLFPPKPKDT L Y I T R E PEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY
RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVK
GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS
LSLSPG X
where X is lysine (K) or absent.

A sixth preferred IgG1 sequence for the CH2 and CH3 domains of the Fc Region-containing molecules of the invention is the L234A/L235A substitution (underlined) that reduces or eliminates effector function, and the M252Y sequence that enhances serum half-life. , the S254T and T256E substitutions (underlined) and the S442C substitution (underlined) to allow the two CH3 domains to covalently bind to each other via a disulfide bond or to allow conjugation of drug moieties. ) and The amino acid sequence of such molecule (SEQ ID NO:203) is:
APE AA GGPSV FLFPPKPKDT L Y I T R E PEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY
RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVK
GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS
L C LSPG X
where X is lysine (K) or absent.

For certain antibodies, diabodies and trivalent binding molecules whose Fc region-containing first and third polypeptide chains are not identical, It is desirable to reduce or prevent homodimerization between the CH2-CH3 domains of the peptide chain. The CH2 and/or CH3 domains of such polypeptide chains need not be identical in sequence and are advantageously modified to facilitate complex formation between the two polypeptide chains. For example, steric hindrance prevents interaction with a similarly mutated domain, pairing the mutated domain with complementary or accommodating mutation engineered domains, i.e., "holes" (e.g., substitution with glycine). As such, amino acid substitutions (preferably with amino acids containing bulky side groups that form "humps", such as tryptophan) can be introduced into the CH2 or CH3 domains. Such mutation sets can be engineered into any pair of polypeptides comprising CH2-CH3 domains that form Fc regions that promote heterodimerization. Methods of protein engineering that select heterodimerization over homodimerization are known in the art, particularly for engineering immunoglobulin-like molecules, and are included herein (e.g., each by reference in its entirety). Ridgway et al. (1996) "'Knobs-Into-Holes' Engineering Of Antibody CH3 Domains For Heavy Chain Heterodimerization," Protein Engr. 9:617-621; Atwell et al. (1997) ", which is incorporated herein. Stable Heterodimers From Remodeling The Domain Interface Of A Homodimer Using A Phage Display Library,” J. Mol. Biol. 270: 26-35; and Xie et al. (2005) “A New Format Of Bispecific Antibody: Highly Efficient Heterodimerization, Expression And Tumor Cell Lysis," J. Immunol. Methods 296:95-101;).

A preferred hump is made by modifying the IgG Fc region to contain the modified T366W. A preferred hole is made by modifying the IgG Fc region to contain modifications T366S, L368A and Y407V. To assist in purifying the hole-bearing third polypeptide chain homodimer from the final bispecific heterodimeric Fc region-containing molecule, the protein A binding sites of the hole-bearing CH2 and CH3 domains of the third polypeptide chain were It is preferably mutated by amino acid substitution at position 435 (H435R). Thus, the hole-bearing third polypeptide chain homodimer does not bind protein A, whereas the bispecific heterodimer retains the ability to bind protein A via the protein A binding site on the first polypeptide chain. do. In another embodiment, the hole-bearing third polypeptide chain can incorporate amino acid substitutions at positions 434 and 435 (N434A/N435K).

A preferred IgG amino acid sequence for the CH2 and CH3 domains of the first polypeptide chain of the Fc Region-containing molecule of the invention is the "hump-bearing" sequence (SEQ ID NO: 109):
APE AA GGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY
RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSL W CLVK
GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS
LSLSPG X
where X is lysine (K) or absent.

the second polypeptide chain of an Fc Region-containing molecule of the invention having two polypeptide chains (or the third polypeptide chain of an Fc Region-containing molecule having three, four, or five polypeptide chains); A preferred IgG amino acid sequence for the CH2 and CH3 domains is the "hole-holding" sequence (SEQ ID NO: 110):
APE AA GGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY
RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSL S C A VK
GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFL V SKL TVDKSRWQQG NVFSCSVMHE ALHN R YTQKS
LSLSPG X
where X is lysine (K) or absent.

As can be seen, the CH2-CH3 domains of SEQ ID NO: 109 and SEQ ID NO: 110 contain substitutions of alanine at position 234 with alanine at 235, thus forming the Fc region, which is the FcγRIA (CD64), reduced (or substantially no) binding to FcγRIIA (CD32A), FcγRIIB (CD32B), FcγRIIIA (CD16a) or FcγRIIIB (CD16b) (compared to the binding shown by the wild-type Fc region (SEQ ID NO: 1)) The present invention also encompasses such CH2-CH3 domains, which contain wild-type alanine residues, alternative and/or additional substitutions that modify the effector function and/or FγR binding activity of the Fc region. .

The present invention also encompasses such CH2-CH3 domains further comprising one or more half-life extending amino acid substitutions. In particular, the invention encompasses such hole-bearing and such hump-bearing CH2-CH3 domains further comprising M252Y/S254T/T256E substitutions.

Exemplary hump-bearing CH2 and CH3 domains containing L234A and L235A substitutions and further containing M252Y, S254T, and T256E substitutions are provided below (SEQ ID NO:204):
APE AA GGPSV FLFPPKPKDT L Y I T R E PEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY
RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSL W CLVK
GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS
LSLSPG X
where X is lysine (K) or absent.

Exemplary hole-holding CH2 and CH3 domains containing L234A and L235A substitutions and further containing M252Y, S254T, and T256E substitutions are provided below (SEQ ID NO:205):
APE AA GGPSV FLFPPKPKDT L Y I T R E PEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY
RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSL S C A VK
GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFL V SKL TVDKSRWQQG NVFSCSVMHE ALHN R YTQKS
LSLSPG X
where X is lysine (K) or absent.

Preferably, the first polypeptide chain has a "hump-bearing" CH2-CH3 sequence, eg, that of SEQ ID NO:109. However, as will be appreciated, a "hole-bearing" CH2-CH3 domain (eg, SEQ ID NO: 110) can be used in the first polypeptide chain, in which case a "hump-bearing" CH2-CH3 domain (eg, SEQ ID NO: 109 ) in the second polypeptide chain of Fc Region-containing molecules of the invention having two polypeptide chains (or in the third polypeptide chain of Fc Region-containing molecules having three, four, or five polypeptide chains chain).

In other embodiments, the present invention relates to PCT Publication Nos. WO 2007/110205; WO 2011/143545; WO 2012/058768; and ADAM9 comprising CH2 and/or CH3 domains engineered to select for heterodimerization over homodimerization using mutations known in the art, such as those disclosed in WO2013/06867 Includes binding molecules.
X. Effector cell binding capacity

As provided herein, the ADAM9 binding molecules of the invention can be engineered to contain a combination of epitope binding sites that recognize a unique set of antigens for a target cell or tissue type. In particular, the present invention provides multispecific ADAM9 binding proteins capable of binding epitopes of ADAM9 and molecules present on the surface of effector cells such as T lymphocytes, natural killer (NK) cells or other mononuclear cells. Regarding molecules. For example, an ADAM9 binding molecule of the invention can be constructed such that it contains an epitope binding site that immunospecifically binds to CD2, CD3, CD8, CD16, the T cell receptor (TCR), or NKG2D. The present invention also relates to trispecific ADAM9 binding molecules capable of binding an epitope of CD3 and an epitope of CD8 (see, eg, PCT Publication No. WO 2015/026894).
A. CD2 binding capacity

In one embodiment, the bispecific, trispecific or multispecific ADAM9 binding molecules of the invention are capable of binding an epitope of ADAM9 and an epitope of CD2. CD2 is a cell adhesion molecule found on the surface of T cells and natural killer (NK) cells. CD2 enhances NK cell cytotoxicity, possibly as a promoter of NK cell nanotube formation (Mace, E.M. et al. (2014) “Cell Biological Steps and Checkpoints in Accessing NK Cell Cytotoxicity,” Immunol. Cell. Biol. 92(3). ):245-255; Comerci, C.J. et al. (2012) "CD2 Promotes Human Natural Killer Cell Membrane Nanotube Formation," PLoS One 7(10):e47664:1-12). Molecules that specifically bind CD2 include the anti-CD2 antibody “Lo-CD2a”.

The amino acid sequence of the VH domain of Lo-CD2a (ATCC Accession No. 11423; SEQ ID NO: 111) is shown below (CDR _H residues are underlined):
EVQLQQSGPE LQRPGASVKL SCKASGYIFT EYYMY WVKQR PKQGLELVG R IDPEDGSIDY VEKFKK KATL
TADTSSNTAY MQLSSLTSED TATYFCAR GK FNYRFAY WGQ GTLVTVSS

The amino acid sequence of the VL domain of Lo-CD2a (ATCC Accession No. 11423; SEQ ID NO: 112) is shown below (CDR _L residues are underlined):
DVVLTQTPPT LLATIGQSVS ISC RSSQSLL HSGNTYLN W LLQRTGQSPQ PLIY LVSKLE S GVPNRFSGS
GSGTDFTLKI SGVEAEDLGV YYC MQFTHYP YT FGAGTKLE LK
B. CD3 binding capacity

In one embodiment, the bispecific, trispecific or multispecific ADAM9 binding molecules of the invention are capable of binding an epitope of ADAM9 and an epitope of CD3. CD3 is a T-cell co-receptor composed of four different chains (Wucherpfennig, K.W. et al. (2010) “Structural Biology Of The T-Cell Receptor: Insights Into Receptor Assembly, Ligand Recognition, And Initiation Of Signaling,” Cold Spring Harb. Perspect. Biol. 2(4):a005140; pages 1-14). In mammals, the complex includes a CD3 gamma chain, a CD3 delta chain, and two CD3 epsilon chains. These chains bind molecules known as T cell receptors (TCRs) to generate activating signals in T lymphocytes. In the absence of CD3, the TCR does not assemble properly and degrades (Thomas, S. et al. (2010) “Molecular Immunology Lessons From Therapeutic T-Cell Receptor Gene Transfer,” Immunology 129(2):170&# 8211;177). CD3 has been found to bind to the membrane of all mature T cells and virtually no other cell types (Janeway, C.A. et al. (2005) In: Immunobiology: The Immune System In Health And Disease," 6th ed. Garland Science Publishing, NY, pp. 214-216; Sun, Z. J. et al. (2001) "Mechanisms Contributing To T Cell Receptor Signaling And Assembly Revealed By The Solution Structure Of An Ectodomain Fragment Of The CD3ε: γ Heterodimer,” Cell 105(7):913-923; Kuhns, M.S. et al. (2006) “Deconstructing The Form And Function Of The TCR/CD3 Complex,” Immunity. 2006 Feb;24(2):133-139 ) Molecules that specifically bind CD3 include the anti-CD3 antibodies "CD3 mAb-1" and "OKT3." The anti-CD3 antibody CD3 mAb-1 is capable of binding non-human primates (eg, cynomolgus monkeys).

The amino acid sequence (SEQ ID NO: 113) of the VH domain of CD3mAb-1 VH(1) is shown below (CDR _H residues are underlined):
EVQLVESGGG LVQPGGSLRL SCAASGFTFS TYAMN WVRQA PGKGLEWVG R IRSKYNNYAT YYADSVKD RF
TISRDDSKNS LYLQMNSLKT EDTAVYYCVR HGNFGNSYVS WFAY WGQGTL VTVSS

The amino acid sequence (SEQ ID NO: 114) of another VH domain of CD3 mAb-1 VH(2) is shown below (CDR _H residues are single underlined; VH domain of CD3 mAb-1 VH(1) (sequence 92) are indicated by double underlining).
EVQLVESGGG LVQPGGSLRL SCAASGFTF N TYAMN WVRQA PGKGLEWV AR IRSKYNNYAT YYADSVKD RF
TISRDDSKNS LYLQMNSLKT EDTAVYYCVR HGNFGNSYVS WFAY WGQGTL VTVSS

The amino acid sequence of the VL domain of CD3 mAb-1 (SEQ ID NO: 115) is shown below (CDR _L residues are underlined):
QAVVTQEPSL TVSPGGTVTL TC RSSTGAVT TSNYAN WVQQ KPGQAPRGLI G GTNKRAP WT PARFSGSLLG
GKAALTITGA QAEDEADYYC ALWYSNLWV F GGGTKLTVLG

The VH domain (SEQ ID NO:) of CD3 mAb-1 VH(1) can be used with the VL domain (SEQ ID NO:) of CD3 mAb-1 to form a functional CD3 binding molecule according to the invention. Similarly, the VH domain (SEQ ID NO:) of CD3 mAb-1 VH(2) can be used with the VL domain (SEQ ID NO:) of CD3 mAb-1 to form a functional CD3 binding molecule according to the invention.

As discussed below, bispecific ADAM9xCD3 binding molecules were produced to illustrate the invention. Variants of CD3 mAb-1 can be used in some of the ADAM9xCD3 constructs. Mutant "CD3 mAb-1 (D65G)" comprises the VL domain of CD3 mAb-1 (SEQ ID NO: 115) and the VH CD3 mAb-1 domain with the D65G substitution (Kabat position 65, at residue 68 of SEQ ID NO: 113). equivalent).

The amino acid sequence (SEQ ID NO: 116) of the VH domain of CD3 mAb-1 (D65G) is shown below (CDR _H residues are underlined and the substituted position (D65G) is double underlined):
EVQLVESGGG LVQPGGSLRL SCAASGFTFS TYAMN WVRQA PGKGLEWVG R IRSKYNNYAT YYADSVKG RF
TISRDDSKNS LYLQMNSLKT EDTAVYYCVR HGNFGNSYVS WFAY WGQGTL VTVSS

Alternatively, affinity variants of CD3 mAb-1 can be incorporated. Variants include low affinity variants designated as "CD3 mAb-1 Low" and variants with a faster off rate designated as "CD3 mAb-1 Fast". The VL domain of CD mAb1 (SEQ ID NO: 115) is common to CD3 mAb-1 Low and CD3 mAb1 Fast and is presented above. The amino acid sequences of the VH domains of each of CD3 mAb-1 Low and CD3 mAb-1 Fast are provided below.

The amino acid sequence (SEQ ID NO: 117) of the VH domain of anti-human CD3 mAb-1 Low is shown below (CDR _H residues are underlined; differences for the VH domain (SEQ ID NO: 113) of CD3 mAb-1 VH(1) is double underlined):
EVQLVESGGG LVQPGGSLRL SCAASGFTFS TYAMN WVRQA PGKGLEWVG R IRSKYNNYAT YYADSVKG RF
TISRDDSKNS LYLQMNSLKT EDTAVYYCVR HGNFGNSYVT WFAY WGQGTL VTVSS

The amino acid sequence of the VH domain of anti-human CD3 mAb-1 Fast (SEQ ID NO: 118) is shown below (CDR _H residues are underlined; compared to the VH domain of CD3 mAb-1 VH(1) (SEQ ID NO: 113): all differences are double underlined):
EVQLVESGGG LVQPGGSLRL SCAASGFTFS TYAMN WVRQA PGKGLEWVG R IRSKYNNYAT YYADSVKG RF
TISRDDSKNS LYLQMNSLKT EDTAVYYCVR HKNFGNSYVT WFAY WGQGTL VTVSS

Another anti-CD3 antibody that can be utilized is the antibody Muromonab-CD3 “OKT3” (Xu et al. (2000) “In Vitro Characterization Of Five Humanized OKT3 Effector Function Variant Antibodies,” Cell. Immunol. 200:16 -26; Norman, D.J. (1995) "Mechanisms Of Action And Overview Of OKT3," Ther. Drug Monit. 17(6):615-620; Canafax, D.M. et al. (1987) "Monoclonal Antilymphocyte Antibody (OKT3) Treatment Of Acute Renal Allograft Rejection,” Pharmacotherapy 7(4):121-124; Swinnen, L.J. et al. (1993) “OKT3 Monoclonal Antibodies Induce Interleukin-6 And Interleukin-10: A Possible Cause Of Lymphoproliferative Disorders Associated With Transplantation,” Curr. Opin. Nephrol. Hypertens. 2(4):670-678).

The amino acid sequence of the VH domain of OKT3 (SEQ ID NO: 119) is shown below (CDR _H residues are underlined):
QVQLQQSGAE LARPGASVKM SCKASGYTFT RYTMH WVKQR PGQGLEWIG Y INPSRGYTNY NQKFKD KATL
TTDKSSSTAY MQLSSLTSED SAVYYCAR YY DDHYCL DYWG QGTTLTVSS

The amino acid sequence of the VL domain of OKT3 (SEQ ID NO: 120) is shown below (CDR _L residues are underlined):
QIVLTQSPAI MSASPGEKVT MTC SASSSVS YMN WYQQKSG TSPKRWIY DT SKLAS GVPAH FRGSGSGTSY
SLTISGMEAE DAATYYC QQW SSNPFTF GSG TKLEINR

Additional anti-CD3 antibodies that can be utilized include, but are not limited to, those described in PCT Publication Nos. WO2008/119566; WO2005/118635.
C. CD8 binding capacity

In one embodiment, a bispecific, trispecific or multispecific ADAM9 binding molecule of the invention is capable of binding an epitope of ADAM9 and an epitope of CD8. CD8 is a T cell co-receptor composed of two different chains that is expressed on cytotoxic T cells (Leahy, DJ, (1995) "A Structural View of CD4 and CD8," FASEB J., 9 :17-25). Activation of CD8 ⁺ T cells is dependent on antigen:major histocompatibility class I (MHC I) molecular complexes placed on the surface of target cells and CD8 and CD8 on the surface of CD8 ⁺ T cells. It has been found to be mediated by co-stimulatory interactions between the T-cell receptor complex (Gao, G., and Jakobsen, B., (2000). "Molecular interactions of coreceptor CD8 and MHC class I: the molecular basis for functional coordination with the T-Cell Receptor". Immunol Today 21: 630–636). Unlike MHC II molecules, which are only expressed by certain immune system cells, MHC I molecules are very ubiquitously expressed. Thus, cytotoxic T cells are capable of binding a wide variety of cell types. Activated cytotoxic T cells mediate cell killing through their release of the cytotoxins perforin, granzyme, and granulysin. Antibodies that specifically bind CD8 include the anti-CD8 antibodies "OKT8" and "TRX2."

The amino acid sequence of the VH domain of OKT8 (SEQ ID NO: 121) is shown below (CDR _H residues are underlined):
QVQLLESGPE LLKPGASVKM SCKA SGYTFT DYNMH WVKQS HGKSLEWIG Y IYPYTGGTGY NQKFKN KATL
TVDSSSSTAY MELRSLTSED SAVYYCARNF RYTYWYFDVW GQGTTVTVSS

The amino acid sequence of the VL domain of OKT8 (SEQ ID NO: 122) is shown below (CDR _L residues are underlined):
DIVMTQSPAS LAVSLGQRAT ISCRASESVD SYDNSLMH WY QQKPGQPPKV LIY LASNLES GVPARFSGSG
SRTDFTLTID PVEADDAATY YC QQNNEDPY T FGGGTKLEI KR

The amino acid sequence of the VH domain of TRX2 (SEQ ID NO: 123) is shown below (CDR _H residues are underlined):
QVQLVESGGG VVQPGRSLRL SCAASGFTFS DFGMN WVRQA PGKGLEWVA L IYYDGSNKFY ADSVKG RFTI
SRDNSKNTLY LQMNSLRAED TAVYYCAK PH YDGYYHFFDS WGQGTLVTVS S

The amino acid sequence of the VL domain of TRX2 (SEQ ID NO: 124) is shown below (CDR _L residues are underlined):
DIQMTQSPSS LSASVGDRVT ITC KGSQDIN NYLA WYQQKP GKAPKLLIY N TDILHT GVPS RFSGSGSGTD
FTFTISSLQP EDIATYYC YQ YNNGYT FGQG TKVEIK
D. CD16 binding capacity

In one embodiment, a multispecific ADAM9 binding molecule of the invention is capable of binding an epitope of ADAM9 and an epitope of CD16. CD16 is the FcγRIIIA receptor. CD16 is expressed by neutrophils, eosinophils, natural killer (NK) cells, and tissue macrophages, which bind aggregated but not monomeric human IgG (Peltz, G.A. et al. (1989) "Human Fc Gamma RIII: Cloning, Expression, And Identification Of The Chromosomal Locus Of Two Fc Receptors For IgG," Proc. Natl. Acad. Sci. (U.S.A.) 86(3):1013-1017; (2014) "NK Cells In Therapy Of Cancer," Crit. Rev. Oncog. 19(1-2):133-141; Miller, J.S. (2013) "Therapeutic Applications: Natural Killer Cells In The Clinic," Hematology Am. Soc. Hematol. Educ. Program. 2013:247-253; Youinou, P. et al. "Autoimmun Rev. 1(1-2):13-19; Peipp, M. et al. (2002) "Bispecific Antibodies Targeting Cancer Cells," Biochem. Soc. Trans. 30(4):507-511). Molecules that specifically bind CD16 include the anti-CD16 antibodies "3G8" and "A9." A humanized A9 antibody is described in PCT Publication No. WO 03/101485.

The amino acid sequence of the VH domain of 3G8 (SEQ ID NO: 125) is shown below (CDR _H residues are underlined):
QVTLKESGPG ILQPSQTLSL TCSFSGFSLR TSGMGVG WIR QPSGKGLEWL A HIWWDDDKR YNPALKS RLT
ISKDTSSNQV FLKIASVDTA DTATYYCAQ I NPAWFAY WGQ GTLVTVSA

The amino acid sequence of the VL domain of 3G8 (SEQ ID NO: 126) is shown below (CDR _L residues are underlined):
DTVLTQSPAS LAVSLGQRAT ISC KASQSVD FDGDSFMN WY QQKPGQPPKL LIY TTSNLES GIPARFSASG
SGTDFTLNIH PVEEEDTATY YC QQSNEDPY T FGGGTKLEI K

The amino acid sequence of the A9 VH domain (SEQ ID NO: 127) is shown below (CDR _H residues are underlined):
QVQLQQSGAE LVRPGTSVKI SCKASGYTFT NYWLG WVKQR PGHGLEWIG D IYPGGGYTNY NEKFKG KATV
TADTSSRTAY VQVRSLTSED SAVYFCAR SA SWYFD VWGAR TTVTVSS

The amino acid sequence of the VL domain of A9 (SEQ ID NO: 128) is shown below (CDR _L residues are underlined):
DIQAVVTQES ALTTSPGETV TLTC RSNTGT VTTSNYAN WV QEKPDHLFTG LIG HTNNRAP GVPARFSGSL
IGDKAALTIT GAQTEDEAIY FC ALWYNNHW V FGGGTKLTVL

Additional anti-CD16 antibodies that can be used include, but are not limited to, those described in PCT Publication Nos. WO 03/101485; and WO 2006/125668.
E. TCR binding capacity

In one embodiment, a bispecific, trispecific or multispecific ADAM9 binding molecule of the invention is capable of binding an epitope of ADAM9 and an epitope of the T cell receptor (TCR). T cell receptors are naturally expressed by CD4 ⁺ or CD8 ⁺ T cells and enable such cells to recognize antigenic peptides bound and presented by class I class II MHC proteins of antigen presenting cells. Recognition of pMHC (peptide – MHC) complexes by the TCR initiates the production of cytokines and the propagation of cell-mediated immune responses that lead to the lysis of antigen-presenting cells (e.g. Armstrong, KM et al. (2008) ". Conformational Changes And Flexibility In T-Cell Receptor Recognition Of Peptide–MHC Complexes,” Biochem. J. 415(Pt 2):183–196; Willemsen, R. (2008) “Selection Of Human Antibody Fragments Directed Against Tumor T-Cell Epitopes For Adoptive T-Cell Therapy,” Cytometry A. 73(11):1093-1099; Beier, KC et al. (2007) “Master Switches Of T-Cell Activation And Differentiation,” Eur. Respir. See J. 29:804-812; Mallone, R. et al. (2005) "Targeting T Lymphocytes For Immune Monitoring And Intervention In Autoimmune Diabetes," Am. J. Ther. 12(6):534–550 ). CD3 is a receptor that binds to the TCR (Thomas, S. et al. (2010) “Molecular Immunology Lessons From Therapeutic T-Cell Receptor Gene Transfer,” Immunology 129(2):170-177; Guy, CS et al. (2009) "Organization Of Proximal Signal Initiation At The TCR: CD3 Complex," Immunol. Rev. 232(1):7-21; St. Clair, EW (Epub 2009 Oct 12) "Novel Targeted Therapies For Autoimmunity," Curr. Opin. Immunol. 21(6):648-657; Baeuerle, PA et al. (Epub 2009 Jun 9) "Bispecific T-Cell Engaging Antibodies For Cancer Therapy," Cancer Res. 69(12):4941-4944 Smith-Garvin, JE et al. (2009) "T Cell Activation," Annu. Rev. Immunol. 27:591-619; Renders, L. et al. (2003) "Engineered CD3 Antibodies For Immunosuppression," Clin. Exp. Immunol. 133(3):307-309).

Molecules that specifically bind to T-cell receptors include the anti-TCR antibody "BMA031" (EP0403156; Kurrle, R. et al. (1989) "BMA 031 – A TCR-Specific Monoclonal Antibody For Clinical Application,” Transplant Proc. 21(1 Pt 1):1017-1019; Nashan, B. et al. (1987) “Fine Specificity Of A Panel Of Antibodies Against The TCR/CD3 Complex,” Transplant Proc. 19( 5):4270-4272; Shearman, C.W. et al. (1991) "Construction, Expression, And Biologic Activity Of Murine/Human Chimeric Antibodies With Specificity For The Human α/β T Cell," J. Immunol. 146(3) :928-935; Shearman, C.W. et al. (1991) "Construction, Expression And Characterization of Humanized Antibodies Directed Against The Human α/β T Cell Receptor," J. Immunol. 147(12):4366-4373; and PCT Publication No. WO 2010/027797).

The amino acid sequence of the VH domain of BMA031 (SEQ ID NO: 129) is shown below (CDR _H residues are underlined):
QVQLVQSGAE VKKPGASVKV SCKASGYKFT SYVMH WVRQA PGQGLEWIG Y INPYNDVTKY NEKFKG RVTI
TADKSTSTAY LQMNSLRSED TAVHYCAR GS YYDYDGFVY W GQGTLVTVSS

The amino acid sequence of the VL domain of BMA031 (SEQ ID NO: 130) is shown below (CDR _L residues are underlined):
EIVLTQSPAT LSLSPGERAT LSC SATSSVS YMH WYQQKPG KAPKRWIY DT SKLAS GVPSR FSGSGSGTEF
TLTISSLQPE DFATYYC QQW SSNPLT FGQG TKLEIK
F. NKG2D binding capacity

In one embodiment, the multispecific ADAM9 binding molecules of the invention are capable of binding an epitope of ADAM9 and an epitope of the NKG2D receptor. The NKG2D receptor is expressed on all human (and other mammalian) natural killer cells (Bauer, S. et al. (1999) “Activation Of NK Cells And T Cells By NKG2D, A Receptor For Stress-Inducible MICA,” Science 285 (5428):727-729; Jamieson, AM et al. (2002) "The Role Of The NKG2D Immunoreceptor In Immune Cell Activation And Natural Killing," Immunity 17(1):19-29) and all CD8 ⁺ T cells. (Groh, V. et al. (2001) "Costimulation Of CD8αβ T Cells By NKG2D Via Engagement By MIC Induced On Virus-Infected Cells," Nat. Immunol. 2(3):255-260; Jamieson (2002) "The Role Of The NKG2D Immunoreceptor In Immune Cell Activation And Natural Killing," Immunity 17(1):19-29). Such binding ligands, particularly those not expressed on normal cells, include the histocompatibility 60 (H60) molecule, the product of retinoic acid early-inducible gene-1 (RAE-1), and murine UL16 binding protein-like transcripts. 1 (MULT1) (Raulet DH (2003) “Roles Of The NKG2D Immunoreceptor And Its Ligands,” Nature Rev. Immunol. 3:781-790; Coudert, JD et al. (2005) “Altered NKG2D Function In NK Cells Induced By Chronic Exposure To Altered NKG2D Ligand-Expressing Tumor Cells,” Blood 106:1711-1717)). Molecules that specifically bind to NKG2D receptors include the anti-NKG2D antibodies "KYK-1.0" and "KYK-2.0" (Kwong, KY et al. (2008) "Generation, Affinity Maturation, And Characterization Of A Human Anti-Human NKG2D Monoclonal Antibody With Dual Antagonistic And Agonistic Activity,” J. Mol. Biol. 384:1143-1156).

The amino acid sequence of the VH domain of KYK-1.0 (SEQ ID NO: 131) is shown below (CDR _H residues are underlined):
EVQLVESGGG VVQPGGSLRL SCAASGFTFS SYGMH WVRQA PGKGLEWVA F IRYDGSNKYY ADSVKG RFTI
SRDNSKNTKY LQMNSLRAED TAVYYCAK DR FGYYLDY WGQ GTLVTVSS

The amino acid sequence of the VL domain of KYK-1.0 (SEQ ID NO: 132) is shown below (CDR _L residues are underlined):
QPVLTQPSSV SVAPGETARI PC GGDDIETK SVH WYQQKPG QAPVLVIY DD DDRPS GIPER FFGSNSGNTA
TLSISRVEAG DEADYYC QVW DDNNDEWV FG GGTQLTVL

The amino acid sequence of the VH domain of KYK-2.0 (SEQ ID NO: 133) is shown below (CDR _H residues are underlined):
QVQLVESGGG LVKPGGSLRL SCAASGFTFS SYGMH WVRQA PGKGLEWVA F IRYDGSNKYY ADSVKG RFTI
SRDNSKNTLY LQMNSLRAED TAVYYCAK DR GLGDGTYFDY WGQGTTVTVS S

The amino acid sequence of the VL domain of KYK-2.0 (SEQ ID NO: 134) is shown below (CDR _L residues are underlined):
QSALTQPASV SGSPGQSITI SC SGSSSNIG NNAVN WYQQL PGKAPKLLIY YDDLLPS GVS DRFSGSKSGT
SAFLAISGLQ SEDEADYYC A AWDDSLNGPV FGGGTKLTVL
XI. Multispecific ADAM9 binding molecules A. ADAM9xCD3 bispecific two-chain diabodies

composed of two covalently linked polypeptide chains using the VL and VH domains of said optimized humanized anti-ADAM9 MAB-A antibody, comprising said optimized humanized VL and VH domains of MAB-A An ADAM9xCD3 bispecific diabody is constructed. The general structure and amino acid sequences of such ADAM9xCD3 bispecific diabodies are presented below.

The first polypeptide chain of one exemplary ADAM9×CD3 bispecific two-chain diabody consists, in the N-terminal to C-terminal direction: the N-terminus; the VL domain of an anti-ADAM9 antibody (e.g., hMAB- A VL(2) (SEQ ID NO: 55); intervening spacer peptide (linker 1: GGGSGGGG (SEQ ID NO: 69)); VH domain of an anti-CD3 antibody (e.g. CD3 mAb1 (D65G) (SEQ ID NO: 116)); containing an intervening spacer peptide (linker 2: GGCGGG (SEQ ID NO:70)); a heterodimer promoting (eg, E-coil) domain (EVAALEK-EVAALEK-EVAALEK-EVAALEK (SEQ ID NO:82)); and the C-terminus.

The second polypeptide chain of such an exemplary ADAM9xCD3 bispecific two-chain diabody consists, in the N-terminal to C-terminal direction: N-terminus; the VL domain of the corresponding anti-CD3 antibody ( For example, a VL domain that together with the VH domain of the first polypeptide chain forms a CD3 binding site, such as the VL domain of CD3 mAb-1 (SEQ ID NO: 115); an intervening spacer peptide (linker 1: GGGSGGGG (SEQ ID NO: 69)); the VH domain of the corresponding anti-ADAM9 antibody (e.g., the VH domain that together with the VL domain of the first polypeptide chain forms the ADAM9 binding site, e.g. hMAB-A VH(2) (SEQ ID NO: 17) an intervening spacer peptide containing a cysteine (linker 2: GGCGGG (SEQ ID NO:70)); a heterodimer promoting (e.g., K-coil) domain (KVAALKE-KVAALKE-KVAALKE-KVAALKE (SEQ ID NO:83)); include.

As presented herein, alternative linkers and/or alternative heterodimer-promoting domains may be utilized in the generation of such diabodies. For example, the first polypeptide chain of another exemplary ADAM9xCD3 bispecific two-chain diabody, in the N-terminal to C-terminal direction: N-terminus; VL domain of anti-ADAM9 antibody; intervening Spacer peptide (linker 1: GGGSGGGG (SEQ ID NO: 69)); VH domain of anti-CD3 antibody or corresponding anti-CD3 antibody; intervening spacer peptide (linker 2: ASTKG (SEQ ID NO: 74)); cysteine-containing heterodimer promoting (eg, K-coil) domains (KVAACKE-KVAALKE-KVAALKE-KVAALKE (SEQ ID NO: 85)); and the C-terminus. The second polypeptide chain of such another exemplary diabody consists, in the N-terminal to C-terminal direction: N-terminus; the corresponding anti-CD3 antibody VL domain; an intervening spacer peptide (linker 1: GGGSGGGG ( VH domain of the corresponding anti-ADAM9 antibody; intervening spacer peptide (linker 2: ASTKG (SEQ ID NO: 74)); heterodimer promoting (e.g., E-coil) domain containing cysteine (EVAACEK-EVAALEK- EVAALEK-EVAALEK (SEQ ID NO: 84)); and the C-terminus.

Representative ADAM9×CD3 bispecific two-chain diabodies (“DART-1”) containing the VH and VL domains of hMAB-A (2.2) and the VH and VL domains of CD3 mAb-1 to build.

The amino acid sequence (SEQ ID NO: 135) of the first polypeptide chain of DART'1 is shown below (hMAb-A VL(2) domain sequence (SEQ ID NO: 55) is underlined; CD3 mAb-1 (D65G) The sequence of the VH domain (SEQ ID NO: 116) is in italics):

The amino acid sequence (SEQ ID NO: 136) of the second polypeptide chain of DART‑1 is shown below (the sequence of the hMAB-A VH(2) domain (SEQ ID NO: 17) is underlined; Sequence (SEQ ID NO: 115) is in italics):

Ｂ．ＡＤＡＭ９×ＣＤ３二重特異的な三本鎖のダイアボディ

B. ADAM9xCD3 bispecific triplex diabodies

One binding site specific for ADAM9 (comprising the humanized/optimized VH and VL domains of hMAB-A) and one binding site specific for CD3 (comprising the VL and VH domains of CD3 mAb1 (D65G)) ADAM9xCD3 diabodies are generated that have triplexes and Fc regions with The diabody is designated "DART-2".

The first polypeptide chain of an exemplary ADAM9×CD3 bispecific three-chain diabodies, in the N-terminal to C-terminal direction: N-terminal; VL domain of anti-ADAM9 antibody (e.g., hMAB-A VL 2) (SEQ ID NO: 55)); intervening spacer peptide (linker 1: GGGSGGGG (SEQ ID NO: 69)); VH domain of CD3 mAb1 (D65G) (SEQ ID NO: 116); intervening spacer peptide (linker 2: ASTKG (sequence cysteine-containing heterodimer promoting (E-coil) domains (EVAACEK-EVAALEK-EVAALEK-EVAALEK (SEQ ID NO: 82)); intervening spacer peptide (linker 3: GGGDKTHTCPPCP (SEQ ID NO: 94)); IgG1 CH2-CH3 domains (SEQ ID NO: 109); and C-terminus. Polynucleotides encoding this polypeptide chain may encode the C-terminal lysine residue of SEQ ID NO: 109 (i.e., X of SEQ ID NO: 109), although, as noted above, this lysine residue is partially expressed. The system can be removed after translation. Accordingly, the present invention provides such lysine residues (i.e., SEQ ID NO: 109 where X is lysine), as well as first polypeptide chains lacking such lysine residues (i.e., SEQ ID NO: 109 where X is absent). ). The amino acid sequence of such first polypeptide chain of DART-2 (SEQ ID NO: 137) is presented below (hMAB-A VL(2) domain sequence (SEQ ID NO: 55) is underlined; CD3 mAb-1 (D65G) VH domain sequence (SEQ ID NO: 116) is in italics):

ここで、Ｘはリジン（Ｋ）であるかまたは存在しない。

where X is lysine (K) or absent.

The second polypeptide chain of an exemplary ADAM9xCD3 bispecific three-chain diabodies, in the N-terminal to C-terminal direction: N-terminal; VL domain of CD3 mAb1 (SEQ ID NO: 115); spacer peptide (linker 1: GGGSGGGG (SEQ ID NO: 69)); VH domain of anti-ADAM9 antibody (e.g., hMAB-A VH(2) (SEQ ID NO: 17)); intervening spacer peptide (linker 2: ASTKG (SEQ ID NO: 17)); 74)); a cysteine-containing heterodimer promoting (K-coil) domain (KVAACKE-KVAALKE-KVAALKE-KVAALKE (SEQ ID NO: 85)); and the C-terminus. The amino acid sequence of such second polypeptide chain of DART-2 (SEQ ID NO: 138) is presented below (hMAB-A VH(2) domain sequence (SEQ ID NO: 17) is underlined; CD3 mAb- 1 VL domain sequence (SEQ ID NO: 115) is in italics):

The third polypeptide chain of an exemplary ADAM9xCD3 bispecific triple-chain diabody, in the N-terminal to C-terminal direction: N-terminus; spacer peptide (DKTHTCPPCP (SEQ ID NO: 93)); hole-bearing IgG1 CH2-CH3 domains (SEQ ID NO: 110); and C-terminus. A polynucleotide encoding this polypeptide chain can encode the C-terminal lysine residue of SEQ ID NO: 110 (i.e., X of SEQ ID NO: 110), although as noted above, this lysine residue is partially It can be removed post-translationally in an expression system. Accordingly, the present invention provides third polypeptide chains comprising such lysine residues (i.e., SEQ ID NO: 110, wherein X is lysine), as well as third polypeptide chains lacking such lysine residues. It includes a polypeptide chain (ie, SEQ ID NO: 110, where X is absent). The amino acid sequence of such a third polypeptide chain (SEQ ID NO: 139) is presented below:
DKTHTCPPCP APEAAGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK
PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK
NQVSLSCAVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLVSKL TVDKSRWQQG NVFSCSVMHE
ALHNRYTQKS LSLSPG X
where X is lysine (K) or absent.

Given the teachings presented herein, different domain orientations, VH domains, VL domains, linkers, and/or heterodimer-promoting domains can be utilized to generate additional ADAM9xCD3 bispecific triple-chain diabodies. can be generated. In particular, the VH and VL domains of different hMAB-A variants can be utilized.
C. ADAM9xCD3xCD8 trivalent binding molecule

One binding site specific for ADAM9, one binding site specific for CD3 (e.g., the VL domain of CD3 mAb-1 (SEQ ID NO: 115), and an anti-CD3 antibody (e.g., CD3 mAb1 ( D65G) and one binding site specific for CD8 (e.g., the VH and VL domains of TRX2 (SEQ ID NOS: 123 and 124, respectively)). "ADAM9xCD3xCD8" binding molecules (comprising parental and/or humanized anti-ADAM9-VL domains and corresponding anti-ADAM9-VH domains, as described above) Such trivalent binding molecules comprise two polypeptides chains (see, eg, FIGS. 6E and 6F), three polypeptide chains (see, eg, FIGS. 6C and 6D), four polypeptide chains (see, eg, FIGS. 6A and 6B), or five can have four polypeptide chains (see, eg, FIG. 5).
XII. Production method

ADAM9 binding molecules of the invention are most preferably produced by recombinant expression of nucleic acid molecules encoding such polypeptides, as is well known in the art.

Polypeptides of the invention can be conveniently prepared using solid-phase peptide synthesis (Merrifield, B. (1986) "Solid Phase Synthesis," Science 232(4748):341-347; Houghten, R.A. (1985) "General Method For The Rapid Solid-Phase Synthesis Of Large Numbers Of Peptides: Specificity Of Antigen-Antibody Interaction At The Level Of Individual Amino Acids," Proc. Natl. Acad. Sci. Ganesan, A. (2006) "Solid-Phase Synthesis In The Twenty-First Century," Mini Rev. Med. Chem. 6(1):3-10).

Alternatively, the antibody can be made recombinantly and expressed using any method known in the art. Antibodies are produced recombinantly by first isolating the antibody produced from a host animal, obtaining the gene sequence, and using the gene sequence to recombinantly express the antibody in a host cell (e.g., CHO cells). can be made to Another method that can be used is to express the antibody sequences in plants (eg tobacco) or transgenic milk. Suitable methods for expressing antibodies recombinantly in plants or milk have been disclosed (see, eg, Peeters et al. (2001) “Production Of Antibodies And Antibody Fragments In Plants,” Vaccine 19:2756; Lonberg, N. et al. (1995) "Human Antibodies From Transgenic Mice," Int. Rev. Immunol 13:65-93; and Pollock et al. (1999) "Transgenic Milk As A Method For The Production Of Recombinant Antibodies," J. (See Immunol Methods 231:147-157). Suitable methods for making derivatives of antibodies, such as humanization, single chains, etc., are known in the art and have been described above. Alternatively, antibodies can be produced recombinantly by phage display technology (e.g., US Pat. Nos. 5,565,332; 5,580,717; 5,733,743). 6,265,150; and Winter, G. et al. (1994) "Making Antibodies By Phage Display Technology," Annu. Rev. Immunol. 12.433-455).

A vector containing a polynucleotide of interest (eg, a polynucleotide encoding a polypeptide chain of an ADAM9 binding molecule of the invention) can be electroporated, calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or other substances. microprojectile bombardment; lipofection; and infection (eg, where the vector is an infectious agent such as vaccinia virus). The choice of vector or polynucleotide to be introduced often depends on host cell characteristics.

Any host cell capable of overexpressing heterologous DNA can be used to express the polypeptide or protein of interest. Non-limiting examples of suitable mammalian host cells include, but are not limited to COS, HeLa, and CHO cells.

The invention includes polypeptides comprising the amino acid sequences of the ADAM9 binding molecules of the invention. Polypeptides of the invention can be made by procedures known in the art. Polypeptides can be produced by proteolytic or other degradation of the antibody, by recombinant methods (ie, single or fusion polypeptides) as described above, or by chemical synthesis. Polypeptides of the antibodies, particularly shorter polypeptides up to about 50 amino acids, are conveniently produced by chemical synthesis. Methods of chemical synthesis are known in the art and commercially available.

The invention includes variants of ADAM9 binding molecules, including functionally equivalent polypeptides that do not significantly affect the properties of such molecules as well as variants with increased or decreased activity. Modification of polypeptides is routine practice in the art and need not be described in detail herein. Examples of modified polypeptides include the use of conservative substitutions of amino acid residues, polypeptides with one or more deletions or additions of amino acids that do not significantly or detrimentally alter their functional activity, or the use of chemical analogs. Amino acid residues that can be conservatively substituted for each other include, but are not limited to: glycine/alanine; serine/threonine; valine/isoleucine/leucine; asparagine/glutamine; and phenylalanine/tyrosine. These peptides include glycosylated and non-glycosylated polypeptides, as well as polypeptides with other post-translational modifications such as glycosylation with different sugars, acetylation, and phosphorylation. Preferably, amino acid substitutions are conservative, ie, the replacement amino acid has similar chemical properties as the original amino acid. Such conservative substitutions are known in the art and examples are provided above. Amino acid modifications can range from changing or modifying one or more amino acids to completing a redesign of a region such as the variable domain. Alterations in the variable domain can alter binding affinity and/or specificity. Other methods of modification include using coupling techniques known in the art including, but not limited to, enzymatic means, oxidative substitution and chelation. Modifications can be used, for example, to attach labels for immunoassays, for example to attach radioactive moieties for radioimmunoassays. Modified polypeptides can be made using procedures established in the art and screened using standard assays known in the art.

The invention includes fusion proteins comprising one or more anti-ADAM9-VL and/or VH of the invention. In one embodiment, fusion polypeptides are provided comprising light chains, heavy chains or both light and heavy chains. In another embodiment, the fusion polypeptide comprises a heterologous immunoglobulin constant region. In another embodiment, the fusion polypeptide comprises the light and heavy chain variable domains of an antibody produced from a publicly deposited hybridoma. For purposes of the present invention, an antibody fusion protein is defined as one or more poly(s) that specifically bind ADAM9 and another amino acid sequence that does not bind in the native molecule, e.g., a heterologous or homologous sequence from another region. Contains a peptide domain.

The invention specifically includes ADAM9 binding molecules (eg, antibodies, diabodies, trivalent binding molecules, etc.) conjugated to diagnostic or therapeutic moieties. For diagnostic purposes, the ADAM9 binding molecules of the invention can be coupled to detectable substances. Such ADAM9 binding molecules are useful for monitoring and/or monitoring the development or progression of disease as part of clinical trial procedures, such as determining the efficacy of a particular therapy. Examples of detectable substances include various enzymes (eg horseradish peroxidase, β-galactosidase, etc.), prosthetic groups (eg avidin/biotin), fluorophores (eg umbelliferone, fluorescein, or phycoerythrin). , luminescent substances (e.g. luminol), bioluminescent substances (e.g. luciferase or aequorin) radioactive substances (e.g. carbon-14, manganese-54, strontium-85 or zinc-65), positron emitting metals, and non-radioactive paramagnetic metal ions. be done. Detectable substances can be coupled or conjugated directly to ADAM9 binding molecules or indirectly through intermediates (eg, linkers) using techniques known in the art.

For therapeutic purposes, the ADAM9 binding molecules of the invention can be conjugated to therapeutic moieties such as cytotoxins, (eg, cytostatic or cytocidal agents), therapeutic agents or radioactive metal ions, eg, alpha emitters. Cytotoxins or cytotoxic agents include any agent that is detrimental to cells, such as Pseudomonas exotoxin, diphtheria toxin, botulinum toxins AF, ricinabrin, saporin, and cytotoxic fragments of such agents. mentioned. A therapeutic agent includes any agent that has a therapeutic effect to treat a disorder, either prophylactically or therapeutically. Such therapeutic agents may be chemotherapeutic agents, protein or polypeptide therapeutic agents, and include therapeutic agents that have a desired biological activity and/or modify a given biological response. . Examples of therapeutic agents include alkylating agents, anti-angiogenic agents, anti-mitotic agents, hormonal therapeutic agents and antibodies useful in treating cell proliferative disorders. A therapeutic moiety can be coupled or conjugated to an ADAM9 binding molecule directly or indirectly through an intermediate (eg, linker) using techniques known in the art.
XIII. Uses of ADAM9-binding molecules of the invention

The invention provides ADAM9 binding molecules (e.g., antibodies, bispecific antibodies, bispecific diabodies, trivalent binding molecules, etc.) of the invention, polypeptides derived from such molecules, such molecules or polypeptides. Compositions, including pharmaceutical compositions, comprising a polynucleotide comprising a sequence encoding a peptide and other agents described herein are encompassed.

As provided herein, the ADAM9-binding molecules of the invention comprising the anti-ADAM9-VL and/or VH domains provided herein have the ability to bind ADAM9 present on the surface of cells. and induce antibody-dependent cell-mediated cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC) and/or redirected cell killing (e.g., redirected T cell toxicity).

Accordingly, ADAM9-binding molecules of the invention comprising anti-ADAM9-VL and/or VH domains provided herein may be used to treat any disease or condition associated with or characterized by ADAM9 expression. have the ability to As previously mentioned, ADAM9 is an onco-embryonic tumor expressed in many hematological and solid malignancies that are associated with high-grade tumors that exhibit poorly differentiated morphology and correlate with poor clinical outcome. ) antigen. Thus, without limitation, the ADAM9-binding molecules of the invention can be used in the diagnosis or treatment of cancers, particularly cancers characterized by ADAM9 expression.

In particular, the ADAM9-binding molecules of the present invention are useful for bladder cancer, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, gastric cancer, head and neck cancer, liver cancer, lymphocytic cancer, non-small cell lung cancer. , bone marrow cancer, ovarian cancer, pancreatic cancer, prostate cancer, renal cell cancer, thyroid cancer, testicular cancer, and uterine cancer.

In a further embodiment, the ADAM-9 binding molecules of the invention are used in non-small cell lung cancer (squamous cell, adenocarcinoma, or undifferentiated large cell carcinoma) and colorectal cancer (adenocarcinoma, gastrointestinal carcinoid tumor, gastrointestinal carcinoid tumor). stromal tumor, primary colonic lymphoma, leiomyosarcoma, melanoma, or squamous cell carcinoma).

The bispecific ADAM9 binding molecules of the invention are useful for tumor cells that express the secondary specificity of such molecules (e.g., CD2, CD3, CD8, CD16, T cell receptor (TCR), NKG2D, etc.). Enhance cancer therapy provided by ADAM9 by promoting redirected killing. Such ADAM9 binding molecules are particularly useful for treating cancer.

In addition to their utility in therapy, the ADAM9 binding molecules of the invention can be detectably labeled and used in cancer diagnosis or imaging of tumors and tumor cells.
XIV. Pharmaceutical composition

Compositions of the present invention include bulk drug compositions useful in the manufacture of pharmaceutical compositions (e.g., impure or non-sterile compositions) and pharmaceutical compositions that can be used in the preparation of unit dosage forms (i.e., dosage to a subject or patient). compositions suitable for administration). Such compositions comprise a prophylactically or therapeutically effective amount of an ADAM9 binding molecule of the invention, or a combination of such agents, and a pharmaceutically acceptable carrier. Preferably, compositions of the invention comprise a prophylactically or therapeutically effective amount of an ADAM9 binding molecule of the invention and a pharmaceutically acceptable carrier. The invention further encompasses pharmaceutical compositions further comprising a second therapeutic antibody (eg, a tumor-specific monoclonal antibody) specific for a particular cancer antigen and a pharmaceutically acceptable carrier.

In specific embodiments, the term "pharmaceutically acceptable" means approved by a federal or US governmental regulatory agency, or approved by the United States Pharmacopoeia or other commonly used pharmaceuticals for use in animals, particularly humans. It means that it is described in a recognized pharmacopoeia. The term "carrier" refers to a diluent, adjuvant (e.g., Freud's adjuvant (complete and incomplete), excipient, or vehicle with which the therapeutic is administered). or mixed together into a unit dosage form and dispensed, for example, as a lyophilized powder or anhydrous concentrate in a hermetically sealed container, such as an ampoule or sachet indicating the quantity of active agent. When administered, they can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline.If the composition is to be administered by injection, an ampoule of sterile water for injection or saline can be dispensed with the ingredients prior to administration. It can be provided so that it can be mixed.

The invention also provides pharmaceutical packs or kits comprising one or more containers filled with an ADAM9 binding molecule of the invention, alone or together with such other pharmaceutically acceptable carriers. In addition, one or more other prophylactic or therapeutic agents useful in treating disease can also be included in the pharmaceutical pack or kit. The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. Such container(s) may optionally be accompanied by a notice, in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice is intended for human administration. reflect authorization by the agency for the manufacture, use or sale of

The invention provides kits that can be used in the above methods. Kits can include any of the ADAM9 binding molecules of the invention. A kit can include in one or more containers one or more other prophylactic and/or therapeutic agents useful for the treatment of cancer.
XV. Dosing method

Compositions of the invention preferably comprise ADAM9 binding molecules (e.g. antibodies, bispecific antibodies, diabodies, trivalent binding molecules, fusion proteins, etc.) or conjugated ADAM9 binding molecules of the invention, or ADAM9 binding molecules. or to treat, prevent, and attenuate one or more symptoms associated with a disease, disorder, or infection by administering to a subject an effective amount of a pharmaceutical composition comprising a conjugated ADAM9-binding molecule of the invention. be able to. In preferred embodiments, such compositions are substantially purified (ie, substantially free of substances that limit their effectiveness or produce undesired side effects). In specific embodiments, the subject is an animal, preferably a non-primate (e.g., bovine, equine, feline, canine, rodent, etc.) or primate (e.g., monkey such as cynomolgus monkey, human, etc.). mammals. In preferred embodiments, the subject is human.

A variety of delivery systems are known, such as, for example, encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing antibodies or fusion proteins, receptor-mediated endocytosis, and the like, and administration of the compositions of the invention. (See, eg, Wu et al. (1987) "Receptor-Mediated In Vitro Gene Transformation By A Soluble DNA Carrier System," J. Biol. Chem. 262:4429-4432). Such as construction of nucleic acids as part of a retrovirus or other vector.

Methods of administering the ADAM9-binding molecules of the invention include, but are not limited to, parenteral administration (e.g., intradermal, intramuscular, intraperitoneal, intravenous and subcutaneous), epidural, and mucosal (e.g., , intranasal and oral routes). In specific embodiments, the ADAM9 binding molecules of the invention are administered intramuscularly, intravenously, or subcutaneously. The compositions may be administered by any convenient route, for example, by infusion or bolus injection, by absorption through epithelial or mucosal linings (eg, oral mucosa, rectal and intestinal mucosa, etc.), and other biologics. can be administered with a therapeutically active agent. Administration can be systemic or local. In addition, pulmonary administration can be employed, eg, through the use of an inhaler or nebulizer, and formulation with an aerosolizing agent. For example, U.S. Pat. Nos. 6,019,968; 5,985,320; 5,985,309; 5,934, each of which is incorporated herein by reference in its entirety. 5,874,064; 5,855,913; 5,290,540; and 4,880,078; WO 97/32572; WO 97/44013; WO 98/31346; and WO 99/66903.

The invention also provides that preparations of the ADAM9 binding molecules of the invention are packaged in sealed containers, such as ampules or sachets, indicating the quantity of the molecule. In one embodiment, such molecules can be provided as dry, sterile, lyophilized powders or anhydrous concentrates in sealed containers, and reconstituted with, for example, water or saline to a concentration suitable for administration to a subject. Preferably, the ADAM9 binding molecules of the invention are supplied as a dry, sterile, lyophilized powder in a closed container.

Lyophilized preparations of the ADAM9-binding molecules of the invention should be stored in their original container at 2° C.-8° C., and the molecules should be stored within 12 hours, preferably within 6 hours, after renaturation. Must be administered within hours, within 3 hours, or within 1 hour. In another embodiment, such molecules are supplied in liquid form in closed containers indicating the amount and concentration of the molecule, fusion protein, or conjugated molecule. Preferably, when such ADAM9 binding molecules are provided in liquid form, they are supplied in a closed container.

As used herein, an “effective amount” of a pharmaceutical composition means, for example, without limitation, reducing symptoms resulting from disease, symptoms of infection (e.g., viral load, fever, pain, sepsis, etc.) or Producing beneficial or desired results, including clinical results such as attenuation of cancer symptoms (e.g., cancer cell proliferation, tumor presence, tumor metastasis, etc.), thereby improving the quality of life of those afflicted with the disease. , reduce the dose of other drugs required to treat the disease, enhance the efficacy of other drug therapies, such as by targeting and/or internalization, slow disease progression, and/or prolong survival of the individual. is sufficient to

An effective amount can be administered in one or more doses. In the context of the present invention, an effective amount of a drug, compound, or pharmaceutical composition is: killing cancer cells and/or reducing cancer cell proliferation and/or eliminating the development of metastases from the primary site of cancer. , a sufficient amount for reduction and/or delay. In some embodiments, an effective amount of a drug, compound, or pharmaceutical composition may or may not be achieved in combination with another drug, compound, or pharmaceutical composition. Thus, an "effective amount" can be administered in the context of administering one or more chemotherapeutic agents, and if the desired result can or will be achieved in combination with one or more other agents. , the single agent can be considered to be administered in an effective amount.

For ADAM9-binding molecules included in the present invention, the dosage administered to a patient is preferably determined based on the body weight (kg) of the recipient subject.

The dosage and frequency of administration of ADAM9-binding molecules of the invention can be reduced or altered by enhancing uptake and tissue penetration of the molecule, eg, by modification, such as lipidation.

The dosage of an ADAM9 binding molecule of the invention administered to a patient can be calculated for use as monotherapy. Alternatively, the molecules can be used in combination with other therapeutic compositions, and the dosage administered to a patient is lower than when said molecules are used as monotherapy.

The pharmaceutical compositions of the present invention can be administered locally to the area in need of treatment; this can be accomplished by, for example, but not limited to, local infusion, injection, or using an implant. The implant can be of porous, non-porous, or jelly-like material, including membranes, such as SILASTIC® membranes, or fibers. Preferably, when administering molecules of the invention, care should be taken to use materials to which the molecules do not absorb.

The compositions of the invention can be delivered in vesicles, in particular liposomes (Langer (1990) "New Methods Of Drug Delivery," Science 249:1527-1533); Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327).

Where the composition of the invention is a nucleic acid encoding an ADAM9 binding molecule of the invention, it may be constructed as part of a suitable nucleic acid expression vector and administered so that it is intracellular, e.g. (see U.S. Pat. No. 4,980,286), or by direct injection, or by use of microparticle bombardment (e.g., gene gun; Biolistic, Dupont), or by lipid or Administering the nucleic acid in vivo by coating it with a cell surface receptor or transducing agent, or by administering it bound to a homeobox-like peptide known to enter the nucleus to encode can promote expression of ADAM9-binding molecules (see, e.g., Joliot et al. (1991) "Antennapedia Homeobox Peptide Regulates Neural Morphogenesis," Proc. Natl. Acad. Sci. (U.S.A.) 88:1864-1868), etc. . Alternatively, the nucleic acid can be introduced intracellularly and integrated into host cell DNA for expression by homologous recombination.

Having generally described the invention, it will be more readily understood by reference to the following examples. The following examples illustrate various methods of composition in diagnostic or therapeutic methods of the invention. The examples are intended to illustrate the scope of the invention and are not limiting in any way.
Example 1
Tumor cell specificity of anti-ADAM9 antibody MAB-A

A murine anti-ADAM9 antibody (designated herein as MAB-A) has been identified that (1) blocks the target protein processing activity of ADAM9; (2) is internalized; and (3) has anti-tumor activity. (See, eg, US Pat. No. 8,361,475). Tumor cell specificity of MAB-A was investigated by IHC. Tumor tissue was contacted with MAB-A (0.4 μg/mL) or isotype control (0.4 μg/mL) and the degree of staining was visualized. MAB-A induced various large cell, squamous, and adenocarcinoma non-small cell lung cancer cell types (Fig. 7A, panels 1-8), breast cancer cells, prostate cancer cells, gastric cancer cells (Fig. 7B, panel 1). ˜6), as well as colon cancer samples (FIG. 7C, panels 1-8). Normal tissue was contacted with MAB-A (1.25 μg/mL) to visualize the extent of staining. As summarized in Table 1 above, MAB-A showed little or no staining of various normal tissues. Note that the concentration of MAB-A used in these studies was nearly three times that used for staining tumor cells. The results of these IHC studies show that MAB-A strongly preferentially binds to tumor cells over normal cells.
Example 2
species cross-reactivity

Binding of MAB-A to human ADAM9 (huADAM9) and cynomolgus monkey ADAM9 (cynoADAM9) was examined. Briefly, 293-FT and CHO-K cells transiently expressing huADAM9, cynoADAM9, an irrelevant antigen, or untransduced parental cells were transfected with MAB-A followed by goat anti-murine-PE secondary antibody. Incubated and analyzed by FACS. As shown in Figures 8A-8B, MAB-A showed strong binding to transiently expressed huADAM9 on both cell types. MAB-A showed poor binding to cynoADAM9. MAB-A did not bind parental cells or cells expressing irrelevant antigens. Similar poor binding to cynoADAM was seen in the ELISA assay.
Example 3
Humanization and initial optimization

Humanization of MAB-A resulted in the humanized VH domain, designated herein as "hMAB-A VH(1)", and the humanized VL domain herein as "hMAB-A VL ( 1)”. The humanized variable domain was then optimized to enhance binding activity and/or remove potentially labile amino acid residues as described in further detail below. A first round of optimization generated three additional humanized VH domain and three further humanized VLs designated herein as "hMAB-A VL(2)", "hMAB-A VL(3)" and "hMAB-A VL(4)" domain and was obtained. In addition, chimeric versions of MAB-A with murine VH and VL domains and human constant regions (“chMAB-A”) were generated. The amino acid sequences of the murine and humanized/optimized VH and VL domains are presented above and alignments are presented in Figures 9A and 9B. Consensus sequences for these humanized/optimized VH and VL domains have been presented previously. When multiple humanized variable domains are generated, a particular anti-ADAM9 antibody (e.g., MAB-A) humanized heavy and light chain variable domains can be used in any combination, with a particular combination of humanized chains being A molecule (eg, an antibody or diabodies) that is referred to by reference to specific VH/VL domains and includes, for example, hMAB-A VH(1) and hMAB-A VL(2) is specifically referred to as "hMAB-A ( 1.2)”.

hMAB-A VH(1) was generated with framework regions from human germline VH3-21 and VH3-64 and hMAB-A VL(1) with framework regions from human germline B3 and L6. was generated. The murine CDRs were retained in these humanized variable domains.

A potential deamidation site was identified in CDR _H 2 (single underlined in Figure 9A) and a potential aspartate isomerization site was identified in CDR _L 1 (singly underlined in Figure 9B). show). Amino acid substitutions at these positions were examined to identify substitutions that eliminated these sites while maintaining binding affinity. Substitution of phenylalanine at position 54 (N54F) of CDR _H 2 (present in hMAB-A VH(2)) and serine at position 28 (D28S) of CDR _L 1 (present in hMAB-A VL(2)) were chosen, where the numbering is according to Kabat. The specified substitutions may be used separately or in combination. Surprisingly, antibodies containing the N54F substitution were found to exhibit approximately a 2-fold increase in affinity to human ADAM9 and slightly improved binding to cynomolgus monkey ADAM9.

Further optimized mutants were generated to minimize the number of lysine residues present in the CDRs. Two lysine residues are present in CDR _H 2 (indicated by double underlining in Figure 9A) and one lysine is present in CDR _L 1 (indicated by double underlining in Figure 9B). Amino acid substitutions at these positions were examined to identify substitutions that maintained binding affinity. An arginine substitution at position 62 (K62R), a glutamine substitution at position 64 (K64Q), and a serine substitution at position 65 (S65G) were selected for CDR _H2 (present in hMAB-A VH(3)), Numbering follows Kabat. An arginine substitution (K24R) at position 24 was chosen for CDR _L 1 (present in hMAB-A VL(3)). The specified substitutions may be used separately or in combination.

Other potentially unstable residues present in the CDRs (indicated by dashed underlining in FIGS. 9A-9B), one methionine residue (M34) within CDR _H 1 at position 34, CDR at position 33 one methionine residue (M33) in _L1 and histidine, glutamic acid, and aspartic acid residues at positions 92 (H93), 93 (E93), and 94 (D94) in CDR _L3 ; Numbering follows Kabat. Amino acid substitutions at these positions were examined to identify substitutions that maintained binding affinity. An isoleucine substitution at position 34 (M34I) was selected for CDR _H 1, and leucine, tyrosine, serine and threonine substitutions at positions 33 (M33L), 92 (H93Y), 93 (E93S), and 94 of CDR _L 3. (D94T), where the numbering is according to Kabat. Each of these positions can be readily substituted in combination with all of the substitutions detailed above to give hMAB-A VH(4) and hMAB-A VL(4), which together pair This produces an antibody that retains some improvement in affinity compared to the parental murine antibody, has a greatly reduced potential for deamidation or oxidation, and has no lysine residues in the CDRs.

Humanized/optimized antibodies hMAB-A (1.1), hMAB-A (2.2), hMAB-A (2.3), hMAB-A (3.3), hMAB-A (4. 4) and the relative binding affinities of chimeric chMAB-A (with murine VH/VL domains) to huADAM were investigated using BIACORE® analysis, in which His-tagged soluble human ADAM9 (“shADAM9-His”, which contains the extracellular portion of human ADAM9 fused to a histidine-containing peptide) was passed over the immobilized antibody-coated surface. Briefly, each antibody was captured on a surface coated with Fab ₂ goat anti-human Fc and then incubated in the presence of different concentrations (6.25-100 nM) of shADAM9-His peptide. Kinetics of binding were determined by BIACORE® assay binding (normalized with a 1:1 Langmuir binding model). The k _a , k _d and K _D calculated from these studies are presented in Table 7. Binding to cynoADAM9 was examined by FACS and ELISA as previously described.

The results of these studies indicate that the humanized/optimized antibody has the same or higher binding affinity for human ADAM9 than the parental murine antibody. In particular, introduction of the N54F mutation into the humanized antibody resulted in improved binding to huADAM9 (i.e. hMAB-A(2.2), hMAB-A(2.3) and hMAB-A(3.3)). was observed. Although this mutation also provides some improvement in binding to cynoADAM9 as determined by FACS and ELISA, these antibodies continue to show poor binding to cynoADAM9. These studies also identified additional substitutions that could be introduced to remove lysine residues from CDRs without reducing affinity. Additional substitutions were identified that had minimal impact on affinity and removed other potentially labile residues.
Example 4
Optimization of binding to non-human primate ADAM9

Random mutagenesis was used to introduce substitutions within the heavy chain CDR _H 2 (Kabat positions 53-58) and CDR _H 3 (Kabat positions 95-100 and 100a-100f) domains of hMAB-A (2.2). did. Mutants were screened to identify clones that had enhanced binding to non-human primate ADAM9 (eg, cynoADAM9) and retained high affinity binding to huADAM9. 48 clones were selected from two independent screens for mutations within CDR _H 3 (Kabat positions 100a-100f). Table 8 presents amino acid sequence alignments of CDR _H 3 Kabat residues 100a-f from hMAB-A (2.2) clones selected for enhanced binding to cynoADAM9 from two independent screens. Additional clone alignments are presented in Table 9. As shown in such tables, similar clones emerged in each experiment and they fell into distinct substitution patterns.

Gly and Ala are the preferred amino acid residues at position 4 (P4) and Leu, Met and Phe are the preferred amino acid residues at position 6 (P6) for all clones examined. Preferred amino acid residues at other positions (eg, position 2 (P2), position 3 (P3) and position 5 (P5)) depend on the amino acid residue found at P1. For clones with a Pro residue at position 1 (P1), Lys and Arg were preferred at P2, Phe and Met at P3, GIy at P4, and Trp or Phe at P5. For clones with Phe, Tyr or Trp at P1, Asn and His were preferred at P2, Ser and His at P3, and Leu at P6. For clones with Ile, Leu or Val at P1, Gly was preferred at P2, Lys at P3, Val at P5, and a hydrophobic at P6. Additionally, as shown in Table 8, for clones with a Thr residue at P1, GIy is preferred at P2, Lys, Met, and Asn are preferred at P3, GIy is preferred at P4, and Val or Thr are preferred at P5. , and Leu and Met were preferred at P6. Additional clones with Asp, Gly, Arg, His, or Ser residues at P1 were also identified at even lower frequencies (see Tables 8 and 9).

A further optimized variant of hMAB-A(2.2), designated hMAB-A(2A.2), was generated using the VH domains of the ten clones shown in Table 9. Binding of selected clones was examined by ELISA assay. Briefly, soluble cynoADAM9 labeled with His-peptide (“cynoADAM9-His”) (1 μg/mL) or with His-peptide was detected using an antibody that bound a histidine-containing peptide and was coated on a microtiter plate. Labeled soluble huADAM9 (1 μg/mL) was captured and serial dilutions of parental hMAB-A (2.2) and 10 CDR _H 3 hMAB-A (2A.2) variants were tested for binding. Binding curves for cynoADAM9 and huADAM9 are presented in Figures 10A and 10B, respectively. hMAB-A(2A.2) variants containing each of the selected VH domains are MAB-A VH(2B), MAB-A VH(2C), MAB-A VH(2D) and MAB-A VH It shows improved binding to cynoADAM9 with (2I), showing the greatest improvement in cynoADAM9 binding while maintaining similar binding to huADAM9 as the parent hMAB-A (2.2) antibody.

Humanized/Further Optimized Antibodies MAB-A VH (2B.2), MAB-A VH (2C.2), MAB-A VH (2D.2) and MAB-A VH (2I.2) and parent The relative binding affinities of hMAB-A (2.2) to huADAM9-His and cynoADAM9-His were investigated using BIACORE® assays essentially as described above. The calculated k _a , k _d and K _D from these studies are presented in Table 10.

Binding studies demonstrated that the four top clones exhibited a 150-550 fold enhancement in binding affinity to cynoADAM9 while maintaining the same high affinity binding to huADAM9 as the parental antibody. hMAB-A (2C.2) and hMAB-A (2I.2) were selected for further study.
Example 5
Immunohistochemical study of antibody hMAB-A (2I.2)

Cell specificity of hMAB-A (2I.2) was examined by IHC. Positive and negative control cells and normal human and cynomolgus tissue were contacted with hMAB-A (2I.2) (2.5 μg/mL) or isotype control (2.5 μg/mL) and the extent of staining visualized. The study results are summarized in Table 11.

An IHC study was also performed to assess binding of humanized/optimized hMAB-A (2I.2) at a concentration of 12.5 μg/mL (5× optimal staining concentration). Positive and negative control cells, normal human tissue, and cynomolgus monkey tissue were employed in this study. The study results are summarized in Table 12.

in binding by hMAB-A (2.2), hMAB-A (2.3), hMAB-A (2C.2), and hMAB-A (2I.2) at 2.5 μg/mL or 5 μg/mL A comparative IHC study was performed to assess differences. Positive and negative control cells, normal human tissue, and cynomolgus monkey tissue were used in this study. The study results are summarized in Table 13.

2.5 μg/mL hMAB-A (2.2), hMAB-A (2.3), hMAB-A (2C.2), and hMAB-A (2I. 2) as well as to evaluate differences in binding by murine MAB-A, further comparative IHC studies were performed. Positive and negative control cells, normal human tissue, and cynomolgus monkey tissue were employed in this study. The study results are summarized in Table 14.

The results therefore show that hMAB-A (2.2) at the optimal concentration showed an overall low level of staining of human hepatocytes and renal tubules, with less reactive staining intensity in hepatocytes and renal tubules in the negative control. / indicates that the frequency was observed. hMAB-A (2.2) showed similarly low levels of staining of cynomolgus monkey hepatocytes and renal tubules at optimal concentrations, with a lower intensity/frequency of reactive staining observed in renal tubules in negative controls.

The results also show that hMAB-A (2C.2) at optimal concentrations showed an overall low level of staining of human hepatocytes and renal tubules, with the reactive staining in hepatocytes and renal tubules observed in the negative control. Indicates low intensity/frequency. hMAB-A (2C.2) showed similar low levels of staining in cynomolgus monkey hepatocytes and renal tubules at optimal concentrations. No further faint findings were observed in hMAB-A (2C.2) cynomolgus monkey lung epithelium, pancreatic islet/epithelium and bladder epithelium in corresponding human tissues; Lower staining intensity/frequency was observed.

The results also show that hMAB-A(2I.2) did not stain human or cynomolgus tissue at optimal concentrations, and +/- bladder transitional cell staining was rare. hMAB-A (2I.2) also showed overall low and frequent staining of human alveolar cells, pancreatic ductal epithelium, renal tubules, bladder transitional cell epithelium at 5× optimal concentration and showed low levels of staining of cynomolgus monkey bronchial epithelium and bladder transitional cell epithelium at 5× optimal concentration. hMAB-A(2I.2) showed an overall favorable IHC profile on the human normal tissues tested and a similar profile on the corresponding cynomolgus monkey tissues.

All publications and patents mentioned in this specification are referred to herein to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference in its entirety. incorporated herein by reference. Although the present invention has been described in relation to specific embodiments thereof, further modifications are possible and this application generally conforms to the principles of the invention and incorporates techniques known or customary in the art to which this invention pertains. It is intended to cover any variations, uses, or adaptations of the invention, including departures from the present disclosure that are within the scope of implementation and applicable to the essential features set forth herein. understood.

Claims (23)

An ADAM9-binding molecule comprising a humanized ADAM9-binding domain, said humanized _ADAM9 -binding domain comprising a heavy chain variable (VH) domain comprising CDR _H 1, CDR _H 2 and CDR _H 3 domains; a light chain variable (VL) domain comprising the 1 domain, the CDR _L 2 domain and the CDR _L 3 domain:
(A) said CDR _H 1 domain, said CDR _H 2 domain, and said CDR _H 3 domain comprise the amino acid sequences of SEQ ID NOS: 8, 35, and 45, respectively, and said CDR _L 1 domain, said CDR _L 2 domain; , and said CDR _L 3 domains comprise the amino acid sequences of SEQ ID NOS: 62, 13, and 14, respectively; or (B) said CDR _H 1 domain, said CDR _H 2 domain, and said CDR _H 3 domains each have the sequence comprising the amino acid sequences of numbers 8, 35, and 37, and said CDR _L 1 domain, said CDR _L 2 domain, and said CDR _L 3 domain comprise the amino acid sequences of SEQ ID NOs: 62, 13, and 14, respectively; or (C) said CDR _H 1 domain, said CDR _H 2 domain, and said CDR _H 3 domain comprise the amino acid sequences of SEQ ID NOs: 8, 35, and 38, respectively, and said CDR _L 1 domain, said CDR _L 2 domain; , and said CDR _L 3 domain comprises the amino acid sequences of SEQ ID NOs: 62, 13 and 14, respectively; or (D) said CDR _H 1 domain, said CDR _H 2 domain, and said CDR _H 3 domain each comprise the sequence comprising the amino acid sequences of numbers 8, 35, and 39, and said CDR _L 1 domain, said CDR _L 2 domain, and said CDR _L 3 domain comprise the amino acid sequences of SEQ ID NOs: 62, 13, and 14, respectively; or (E) said CDR _H 1 domain, said CDR _H 2 domain, and said CDR _H 3 domain comprise the amino acid sequences of SEQ ID NOs: 8, 35, and 40, respectively; and said CDR _L 1 domain, said CDR _L 2 domain; and said CDR _L 3 domains comprise the amino acid sequences of SEQ ID NOs: 62, 13 and 14, respectively; or (F) said CDR _H 1 domain, said CDR _H 2 domain, and said CDR _H 3 domains each comprise the sequence comprising the amino acid sequences of numbers 8, 35, and 41, and said CDR _L 1 domain, said CDR _L 2 domain, and said CDR _L 3 domain comprise the amino acid sequences of SEQ ID NOs: 62, 13, and 14, respectively; or (G) said CDR _H 1 domain, said CDR _H 2 domain, and said CDR _H 3 domain are amino acids of SEQ ID NOs: 8, 35, and 42, respectively and wherein said CDR _L 1 domain, said CDR _L 2 domain, and said CDR _L 3 domain comprise the amino acid sequences of SEQ ID NOs: 62, 13, and 14, respectively; or (H) said CDR _H 1 domain, Said CDR _H 2 domain and said CDR _H 3 domain comprise the amino acid sequences of SEQ ID NOs: 8, 35 and 43, respectively, and said CDR _L 1 domain, said CDR _L 2 domain and said CDR _L 3 domain comprise: or (I) said CDR _H 1 domain, said CDR _H 2 domain, and said CDR _H 3 domain are amino acids of SEQ ID NOs: 8, 35, and 44, respectively and wherein said CDR _L 1 domain, said CDR _L 2 domain, and said CDR _L 3 domain comprise the amino acid sequences of SEQ ID NOs: 62, 13, and 14, respectively; or (J) said CDR _H 1 domain, Said CDR _H 2 domain and said CDR _H 3 domain comprise the amino acid sequences of SEQ ID NOs: 8, 35 and 46, respectively, and said CDR _L 1 domain, said CDR _L 2 domain and said CDR _L 3 domain comprise: or (K) said CDR _H 1 domain, said CDR _H 2 domain, and said CDR _H 3 domain are amino acids of SEQ ID NOs: 8, 35, and 10, respectively and wherein said CDR _L 1 domain, said CDR _L 2 domain, and said CDR _L 3 domain comprise the amino acid sequences of SEQ ID NOS: 62, 13, and 14, respectively; or (L) said CDR _H 1 domain, Said CDR _H 2 domain and said CDR _H 3 domain comprise the amino acid sequences of SEQ ID NOs: 8, 35 and 10, respectively, and said CDR _L 1 domain, said CDR _L 2 domain and said CDR _L 3 domain comprise: or (M) said CDR _H 1 domain, said CDR _H 2 domain, and said CDR _H 3 domain are amino acids of SEQ ID NOs: 8, 36, and 10, respectively and wherein said CDR _L 1 domain, said CDR _L 2 domain, and said CDR _L 3 domain are SEQ ID NOs: 63, 13, and 1, respectively or (N) said CDR _H 1 domain, said CDR _H 2 domain, and said CDR _H 3 domain comprise the amino acid sequences of SEQ ID NOS: 34, 36, and 10, respectively; and said CDR _L 1 domain, said CDR _L 2 domain, and said CDR _L 3 domain comprise the amino acid sequences of SEQ ID NOs: 64, 13, and 65, respectively;
ADAM9 binding molecule. wherein said humanized ADAM9 binding domain:
(A) (1) a CDR _H 1 domain comprising the amino acid sequence of SEQ ID NO:8;
(2) a CDR _H 2 domain comprising the amino acid sequence of SEQ ID NO: 35; and (3) a CDR _H 3 comprising the amino acid sequence of SEQ ID NO: 37, 38, 39, 40, 41, 42, 43, 44, 45, or 46. domain;
and (B) (1) a CDR _L 1 domain comprising the amino acid sequence of SEQ ID NO:62;
(2) a CDR _L 2 domain comprising the amino acid sequence of SEQ ID NO:13; and (3) a CDR _L 3 domain comprising the amino acid sequence of SEQ ID NO:14. wherein said humanized ADAM9 binding domain:
(A) (1) a CDR _H 1 domain comprising the amino acid sequence SYWMH (SEQ ID NO: 8);
(2) a CDR _H 2 domain comprising the amino acid sequence EIIPIFGHTNYNEKFKS (SEQ ID NO: 35); and (3) a CDR _H 3 domain comprising the amino acid sequence GGYYYYPRQGFLDY (SEQ ID NO: 45);
and (B) (1) a CDR _L 1 domain comprising the amino acid sequence KASQSVDYSGDSYMN (SEQ ID NO: 62);
(2) a CDR _L 2 domain comprising the amino acid sequence AASDLES (SEQ ID NO: 13); and (3) a CDR _L 3 domain comprising the amino acid sequence QQSHEDPFT (SEQ ID NO: 14). wherein said humanized ADAM9 binding domain:
(A) a VH domain comprising the amino acid sequence of SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, or SEQ ID NO:29 and (B) the ADAM9-binding molecule of any one of claims 1-3, comprising a VL domain comprising the amino acid sequence of SEQ ID NO:55. wherein said humanized ADAM9 binding domain:
(A) a VH domain comprising the amino acid sequence of SEQ ID NO: 17; and a VL domain comprising the amino acid sequence of SEQ ID NO: 55; or (B) a VH domain comprising the amino acid sequence of SEQ ID NO: 17; or (C) a VH domain comprising the amino acid sequence of SEQ ID NO: 18; and a VL domain comprising the amino acid sequence of SEQ ID NO: 56; or (D) a VH domain comprising the amino acid sequence of SEQ ID NO: 19; a VL domain comprising an amino acid sequence;
The ADAM9-binding molecule of any one of claims 1-3, comprising: 5. The ADAM9 binding molecule of claim 4, wherein said humanized ADAM9 binding domain comprises the VH domain of SEQ ID NO:28. The ADAM9-binding molecule of any one of claims 1-6, wherein said molecule is a monospecific ADAM9-binding antibody or an ADAM9-binding fragment thereof. The ADAM9-binding molecule of any one of claims 1-6, wherein said molecule is a bispecific antibody. The ADAM9-binding molecule of any one of claims 1-6, wherein said molecule is a diabody and said diabody is a covalently linked complex comprising 2, 3, 4 or 5 polypeptide chains. . 7. Any one of claims 1-6, wherein said molecule is a trivalent binding molecule, said trivalent binding molecule being a covalently linked complex comprising 3, 4, 5 or more polypeptide chains. ADAM9 binding molecules as described. 11. The ADAM9 binding molecule of any one of claims 7-10, wherein said ADAM9 binding molecule comprises an Fc region selected from the group consisting of IgGl, IgG2, IgG3 and IgG4. wherein said Fc region is:
(a) reduces the affinity of the variant Fc region for FcγRs compared to that displayed by the wild-type Fc region; and/or (b) reduces the affinity of said ADAM9 binding molecule compared to a molecule comprising a wild-type Fc region 12. The ADAM9 binding molecule of claim 11, which is a variant Fc region comprising one or more amino acid modifications relative to the wild-type Fc region that enhance serum half-life. wherein said one or more amino acid modifications that reduce said affinity of said variant Fc region for FcγRs are:
(A) L234A;
(B) L235A; or (C) L234A and L235A
including
13. The ADAM9 binding molecule of claim 12, wherein the numbering follows the EU index. wherein said one or more amino acid modifications that enhance said serum half-life of said ADAM9-binding molecule are:
(A) M252Y;
(B) M252Y and S254T;
(C) M252Y and T256E;
(D) M252Y, S254T and T256E; or (E) K288D and H435K
includes;
14. The ADAM9 binding molecule of claim 12 or 13, wherein the numbering follows the EU index. 15. The ADAM9-binding molecule of any one of claims 1-14, wherein said molecule further comprises a human IgG CLK domain having the amino acid sequence of SEQ ID NO:101. (a) said CDR _H 1 domain, said CDR _H 2 domain, and said CDR _H 3 domain comprise the amino acid sequences of SEQ ID NOs: 8, 35, and 45, respectively; and said CDR _L 1 domain, said CDR _L 2 domain; , and said CDR _L 3 domains comprise the amino acid sequences of SEQ ID NOs: 62, 13, and 14, respectively;
(b) the molecule comprises an IgG1 Fc region, wherein the Fc region is a variant Fc region comprising the M252Y, S254T and T256E amino acid modifications, numbering according to the EU index;
16. The ADAM9 binding molecule of claim 15. The molecule is bispecific and comprises an epitope binding site capable of immunospecific binding to an epitope of ADAM9 and an epitope binding site capable of immunospecific binding to an epitope of the molecule present on the surface of effector cells. The ADAM9 binding molecule of any one of claims 1-9 or 11-16, comprising. wherein said molecule comprises two epitope binding sites capable of immunospecific binding to an epitope of ADAM9 and two epitope binding sites capable of immunospecific binding to an epitope of the molecule present on the surface of effector cells. 18. The ADAM9 binding molecule of Item 17. said molecule is trispecific and:
(a) an epitope binding site capable of immunospecific binding to an epitope of ADAM9;
(b) an epitope binding site capable of immunospecific binding to an epitope of the first molecule present on the surface of the effector cell;
(c) an epitope binding site capable of immunospecific binding to an epitope of a second molecule present on the surface of an effector cell;
The ADAM9-binding molecule of any one of claims 1-6 or 10-16, comprising. The ADAM9 binding molecule of any one of claims 17-19, wherein said molecule present on said surface of effector cells is CD2, CD3, CD8, TCR or NKG2D. A pharmaceutical composition comprising an effective amount of the ADAM9 binding molecule of any one of claims 1-20 and a pharmaceutically acceptable carrier, excipient or diluent. ADAM9-binding molecule according to any one of claims 1 to 20 or according to claim 21 in the manufacture of a medicament for the treatment of a disease or condition associated with or characterized by ADAM9 expression Use of the described pharmaceutical composition. said disease or condition associated with or characterized by expression of ADAM9 is bladder cancer, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, stomach cancer, head and neck cancer , liver cancer, non-small cell lung cancer, bone marrow cancer, ovarian cancer, pancreatic cancer, prostate cancer, renal cell cancer, thyroid cancer, testicular cancer, and uterine cancer 23. Use according to claim 22, which is cancer.

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